The Impact of Androgen Receptors on ER+ Breast Cancer Microenvironments.

Background: Estrogen receptor positive breast cancer (ER+ BC) is the most frequently diagnosed subtype of BC with consequent highest mortality. For patients with ER+ BC, standard of care treatments are endocrine therapies targeting ER expression and activity or aromatase inhibitors that block synthesis of estrogens from adrenal androgens. However, it is estimated that nearly 33% of all patients with ER+ BC will relapse within 15 years, underscoring the need for additional therapeutic options. We have demonstrated a role for androgen receptors (AR) in promoting tumor progression and therapy resistance. AR has also been shown to be a negative regulator of immune cell function that inhibits anti-tumor immunity in prostate cancer and melanoma models. Preclinical data from our lab resulted in a Phase II neoadjuvant clinical trial in women with large ER+/AR+/HER2- BC (NCT02955394) randomized to either the ER degrader fulvestrant alone or combined with an AR antagonist, enzalutamide (combo). Hypothesis: We postulate that in addition to directly targeting BC cells, AR inhibition also shapes the tumor microenvironment to a pro-inflammatory anti-tumor state.

Methods: From the clinical trial we performed Polaris multiplex protein analyses to identify changes in immune cell populations and tertiary lymphoid structures (TLS). AR-regulated immune-modulatory factors such as Gross Cystic Disease Fluid Protein 15 (GCDF-15), also known as prolactin-inducible protein (PIP), and growth differentiation factor 15 (GDF-15) were measured in patient plasma by ELISA. To identify direct effects of androgens on T cells, we stimulated AR directly in primary human T cells from healthy postmenopausal women or indirectly (via incubation of primary T cells in conditioned media from AR stimulated BC cells) and measured changes in T cell function, differentiation, and exhaustion using established markers (CD3, CD4, CD8, CD45RA, CD45RO, CD27, CD107a, IFNγ, TNFα, GzmB, Tbet, and Eomes) via flow cytometry analysis.

Results: Tumors treated from patients in the combo arm uniquely showed 1) increases in transcripts of many pro-inflammatory pathways, 2) decreases in multiple inhibitory immune cell populations (T regulatory cells, tumor associated macrophages, and myeloid-derived suppressor cells), and 3) increased TLS formation. Additionally, in vitro stimulation of BC cells with androgens results in the expression and secretion of immune-modulatory proteins PIP, GDF-15, and zinc-alpha-2-glycoprotein (ZAG). Notably, using our clinical trial samples we show decreased secreted PIP and GDF-15 over time with treatment.

Conclusion: AR inhibition may “reawaken” the immune microenvironment in ER+ BCs—which are typically immunologically cold—and may render ER+ BC responsive to immune checkpoint inhibitors to improve patient outcomes.