Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

Abstract

Introduction: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.

Methods: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR.

Results: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis.

Conclusions: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Figure 1

Women with tumors having a higher AR:ER ratio have a shorter disease-free and disease-specific overall survival as compared with patients with lower AR:ER ratio. Immunohistochemistry for androgen receptor (AR) and estrogen receptor (ER) were performed on formalin-fixed paraffin-embedded sections of primary breast cancers. Slides were scored for the percent of positive nuclear staining for AR and ER. Ratios were calculated to determine the best cutoff point for analysis. For (A) to (D) women are divided into two groups: those with AR:ER ratios <2.0 (blue squares) and those with AR:ER ratios ≥2.0 (red circles). The number of patients at risk at each time point is reflective of the number of patients censored due to no further follow-up data at each time point (underneath). Kaplan–Meier survival curve for: (A) disease-free survival (DFS) for all patients; (B) disease-specific survival (DSS) overall for all patients; (C) DFS for patients who failed while on tamoxifen therapy; (D) DSS overall for patients who failed while on tamoxifen therapy; and (E) representative images of AR and ER staining from the two groups (400× magnification).

Figure 2

Enzalutamide abrogates androgen mediated proliferation in estrogen receptor-positive breast cancer cells. (A) Baseline levels of androgen receptor (AR) and estrogen receptor (ER) alpha protein in whole cell lysates from ER-positive (MCF7, BCK4, T47D and ZR-75-1) and ER-negative (MDA-MB-453) breast cancer and prostate (LNCaP) cancer cell lines. (B) AR protein levels in MCF7 cells plated in charcoal-stripped serum-containing media for 48 hours prior to treatment with vehicle control, 10 nM 5-alpha-dihydrotestosterone (DHT), 10 μM enzalutamide (Enza) or a combination of DHT and Enza for 48 hours. (C) MCF7 and (D) BCK4 breast cancer cells, both ER + AR+, were treated with vehicle control, 10 nM DHT, 10 μM Enza or a combination of DHT and Enza, and MTS proliferation assays were performed. Error bars represent standard error of the mean. *P < 0.05, ***P < 0.001 for DHT versus DHT + Enza, analysis of variance with Bonferroni’s multiple comparison test correction. (E) AR levels in cytosolic and nuclear fractions of MCF7 cells treated with vehicle, 10 nM DHT, 10 μM enzalutamide or DHT + Enza for 3 hours.

Figure 3

Enzalutamide inhibits androgen-stimulated growth of MCF7 tumors in vivo. MCF7-TGL cells stably expressing luciferase were implanted orthotopically in the mammary gland of NOD/SCID ovariectomized female mice with a 5-alpha-dihydrotestosterone (DHT) pellet implanted subcutaneously. Mice were matched into two groups based on tumor volume (day -2) and treatment with either control chow (DHT) or chow containing 50 mg/kg enzalutamide (DHT + Enza) begun (day 0, indicated by arrow), and the tumor burden was measured by whole-body luminescence. (A) Mean total flux of all mice in each of the treatment groups. Error bar represents standard error of the mean. *P < 0.05, Wilcoxon rank sum. (B) Total luminescent flux is shown for all individual mice at the day of matching (day -2) and at the final imaging day (day 19). *P < 0.05, Wilcoxon rank sum. (C) Mice were injected with bromodeoxyuridine (BrdU) 2 hours prior to sacrifice and BrdU immunohistochemistry was performed on formalin-fixed paraffin-embedded tumor sections and quantified. *P < 0.05, Student’s t test. Representative images of BrdU staining (400× magnification) and quantification. (D) Quantification of apoptotic cells as measured by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining with representative images below (400× magnification). *P < 0.05, Student’s t test. (E) Quantification of nuclear AR staining and representative images (400× magnification). ***P < 0.001, Wilcoxon rank sum.

Figure 4

Enzalutamide inhibits androgen-stimulated growth of MDA-MB-453 tumors. MDA-MB-453 cells were injected orthotopically in the mammary gland of female NOD-SCID-IL2Rgc^-/- mice. Three groups had a 5-alpha-dihydrotestosterone (DHT) pellet implanted subcutaneously and one group had no pellet (Vehicle). Once tumors reached an average size of 100 mm³ (green arrow), mice were given either enzalutamide (Enza, 10 mg/kg) or vehicle (Vehicle and DHT groups) by daily oral gavage. Another group was given a higher dose of Enza (25 mg/kg) by oral gavage when tumors reached an average size of 400 mm³ (blue arrow). (A) Tumor volume was measured weekly by caliper. Error bars represent standard error of the mean. *P < 0.05, **P < 0.01 for DHT versus DHT + Enza (10 mg/kg), Wilcoxon rank sum. (B) Tumors were excised and weighed at the end of the experiment. (C) Tumor sections stained for cleaved caspase 3 were quantified (left) and representative images shown (right) (200× magnification). *P < 0.05, **P < 0.01, ***P < 0.001, analysis of variance with Bonferroni’s multiple comparison test correction. (D) Nuclear androgen receptor staining was quantified (left) and representative images (400× magnification) are shown (right). *P < 0.05, ***P < 0.001, Kruskal–Wallis with Dunn’s multiple comparison test correction.

Figure 5

Enzalutamide inhibits estradiol-mediated proliferation of breast cancer cells, while bicalutamide does not. MTS proliferation assays were performed on (A) MCF7s cells and (B) BCK4 cells treated with vehicle control, 10 nM estradiol (E2), 10 μM enzalutamide (Enza) or a combination of E2 and Enza. Error bars represent standard error of the mean (SEM). **P < 0.01, ***P < 0.001 for E2 versus E2 + Enza, analysis of variance (ANOVA) with Bonferroni’s multiple comparison test correction. (C) MCF7 cells were treated for 48 hours with treatments as above and real-time polymerase chain reaction (PCR) was performed for estrogen-responsive genes, progesterone receptor (PR) and stromal cell-derived factor 1 (SDF-1, also known as CXCL12). Each gene is normalized to 18S and shown relative to vehicle. *P < 0.05, ***P < 0.001, Student’s t test. MCF7 cells were treated with vehicle control, (D) 1 μM bicalutamide, 10 nM 5-alpha-dihydrotestosterone (DHT) and DHT + bicalutamide or (E) with 10 nM E2 and E2 + bicalutamide. **P < 0.01, ***P < 0.001 for DHT versus DHT + bicalutamide, or E2 versus E2 + bicalutamide, ANOVA with Bonferroni’s multiple comparison test correction. (F) MCF7 cells treated for 48 hours with vehicle, 1 μM bicalutamide, 10 nM E2 and E2 + bicalutamide and real-time PCR performed for PR and SDF-1. *P < 0.05, ***P < 0.001, Student’s t test. Error bars represent SEM, Student’s t test (all analyses).

Figure 6

Enzalutamide inhibits estrogen-stimulated growth of MCF7 tumors as effectively as tamoxifen. MCF7-TGL cells stably expressing luciferase were implanted orthotopically in the mammary gland of ovariectomized female nude mice. All mice had an estradiol (E2) pellet implanted subcutaneously and were given either control chow (E2), control chow plus a tamoxifen pellet implanted subcutaneously (E2 + Tam) or chow containing 50 mg/kg enzalutamide (E2 + Enza). The tumor burden was measured by whole-body luminescence. (A) Mean total flux. Mice were matched on day -3 and treatment began on day 0 (arrow). *P < 0.05, analysis of variance (ANOVA) with Bonferroni’s multiple comparison test correction. (B) The total luminescent flux is shown for individual mice on the day of matching (day -3) and of final imaging (day 11). *P < 0.05, ANOVA with Bonferroni’s multiple comparison test correction. (C) Images of luminescent signal in the two treatment groups at time of matching (day -3) and the final day of imaging (day 11). (D) Mice were injected with bromodeoxyuridine (BrdU) 2 hours prior to sacrifice and immunohistochemistry for BrdU was performed on tumor sections and quantified using imageJ (National Institutes of Health, Bethesda, MD, USA). Representative images of BrdU staining (left, 400× magnification) and quantification (right). **P < 0.01 for E2 versus E2 + Tam, ***P < 0.001 for E2 versus E2 + Enza, ANOVA with Bonferroni’s multiple comparison test correction.