Abstract IA18: Targeting the androgen receptor in multiple breast cancer subtypes
Authors: D'Amato Nicholas C. , Cochrane Dawn R. , Barton Valerie N. , Bolin Liu , Edgerton Susan M. , Bernales Sebastian , Gu Haihua , Thor Ann D. , Richer Jennifer K. 
Published: May 2015

Abstract
The androgen receptor (AR) is even more widely expressed in breast cancer (BC) than estrogen receptor alpha (ER) or progesterone receptor (PR), and recently AR has emerged as a useful marker to refine classification of breast cancer (BC). However, we still understand relatively little about the specific effects of AR or its potential as a therapeutic target in the different subtypes of BC. Our data from clinical samples suggests that some ER+ tumors can switch from growth driven by estrogens to growth driven by adrenal androgens, particularly when the estradiol (E2)/ER pathway is inhibited by standard ER-directed endocrine treatments. Indeed, we have evidence showing that a high ratio of AR to ER (≥2.0) leads to a &gt; 4 fold increased risk for relapse while on tamoxifen (HR=4.43, P&lt;0.0001) and is a strong independent predictor of disease-free survival (HR=4.04, P=0.002). Since approximately 30% of metastatic ER+ tumors exhibit de novo resistance to standard endocrine therapy and all patients with metastatic disease ultimately progress with acquired resistance, targeting AR in ER+ BC may be clinically useful. Thus, clinical trials evaluating the role of androgen receptor inhibition in BC are underway.
Importantly, we find that new generation anti-androgens that block AR DNA binding affect BC differently than older generation anti-androgens (which allow DNA binding), leading to new insights into the role of AR. When AR is excluded from the nucleus, both androgen- and estradiol (E2)-stimulated proliferation is inhibited in ER+ breast cancers, suggesting that AR plays a previously-unknown role in E2-mediated ER activity. Both enzalutamide (ENZ) and bicalutamide inhibited androgen-mediated proliferation of breast cancer lines in vitro. Interestingly, ENZ uniquely inhibited E2-mediated proliferation of ER.+/AR+ breast cancer cells in vitro, and also inhibited E2-driven tumor growth as effectively as tamoxifen in vivo. When opposing androgen-stimulated tumor growth in ER+ BC in vivo models, ENZ inhibited proliferation and increased apoptosis, while in ER- models it increased apoptosis but did not alter proliferation. When opposing E2-stimulated tumor growth in vivo ENZ decreased proliferation but did not increase apoptosis.
In Her2+ but non-amplified, BC models the AR inhibitor ENZ, inhibited proliferation as well or better than trastuzumab (TRAS), whereas TRAS showed a greater inhibitory effect than ENZ in Her2 amplified lines. In all models tested, the combination of ENZ and TRAS inhibited proliferation more effectively than either agent alone. In TRAS resistant lines, addition of ENZ to TRAS significantly inhibited proliferation. Liganded AR upregulates Her3 in some Her2+ BC lines; however, in other Her2+ lines, Her3 is not increased by androgen stimulation, yet anti-androgens still inhibit proliferation. Our data suggest that the AR and Her2 pathways are linked in ways not previously understood and that there are novel Her3-independent mechanisms whereby AR impacts Her2+BC.
A subset of TNBC express AR and knock down of AR using lentiviral shRNAs significantly reduced proliferation in multiple TNBC lines. Propidium iodide cell cycle analysis demonstrated that knockdown of AR in TNBC decreased the percentage of cells in G2/M. Lastly, inhibition of AR with ENZ, blocked ligand-mediated nuclear translocation of AR as assessed by nuclear and cytosolic fractionation and significantly decreased proliferation of TNBC in vitro.
In summary, our studies demonstrate that AR plays varying roles in the different BC subtypes and will serve to guide appropriate combinations with AR inhibitor and existing therapeutic strategies. Further, we find that assessment of the amount of AR relative to ER may be informative.
Citation Format: Nicholas C. D'Amato, Dawn R. Cochrane, Valerie N. Barton, Liu Bolin, Susan M. Edgerton, Sebastian Bernales, Haihua Gu, Ann D. Thor, Jennifer K. Richer. Targeting the androgen receptor in multiple breast cancer subtypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA18.