SUN-289 - AR and ER Context-Dependent Dimerization in Breast Cancer

Background: Estrogen receptor positive (ER+) breast cancer is the most common type of breast cancer, representing about 70% of all primary tumors. Although ER is the primary driver of breast cancer growth, androgen receptors (AR) are widely expressed in over 90% of ER+ breast cancers. How AR may influence ER action during breast cancer progression remains controversial since both AR agonists and antagonists have shown some benefit in clinical trials. Previously, we published that AR antagonists reduce estradiol (E2)-mediated ER chromatin binding at 50% of binding sites. Conversely, E2 induced AR binding at unique sites in an ER-dependent manner compared to dihydrotestosterone (DHT)-induced binding sites. Lastly, we published that AR and ER interact upon E2 treatment, as detected by proximity ligation assay. Hypothesis: We postulate that AR and ER can interact and have reciprocal effects on chromatin binding and transcriptional activity. These interactions may occur by stabilizing a monomer or homodimer binding to DNA, or through the formation of ER/AR heterodimers, particularly in the context of stress encountered during cancer progression and treatment, when AR levels are increased.

Methods: Using the NanoBiT protein-protein interaction system in HEK293T cells, which lack endogenous AR, ER, and progesterone receptor (PR) expression, we evaluated the formation of AR and ER monomers, dimers and heterodimers. We investigated how different ER and AR agonists and antagonists at various doses affect homo- or heterodimerization and how different types of cellular stress affect these interactions. We also examined the effects of AR inhibition with enzalutamide or seviteronel on growth in soft agar using the UCD4 cell line derived from a patient with a D538G ER-mutant breast cancer that expresses increased levels of AR upon anchorage independent growth. Lastly, we will analyze how AR affects E2/ER-mediated transcriptional regulation of PR and conversely, how E2/ER affects AR regulation of secreted proteins that affect the tumor immune microenvironment.

Results: Our NanoBiT assay results demonstrate that, as expected, E2 stimulates ER homodimers and DHT stimulates AR homodimers. Excitingly, we find that E2 stimulates formation of AR homodimers about 50% as efficiently as with DHT. Functionally, we find that AR inhibition of ER mutant UCD4 cells in soft agar conditions showed significantly decreased colony number and size following treatment with either enzalutamide or seviteronel. Treatment with enzalutamide and seviteronel both significantly decreased the average area of the colonies by two-fold, while total colony number was decreased three-fold or more.

Conclusions: Our research will help determine how AR functions in ER+ breast cancers during progression to metastatic disease and resistance to current treatments such as aromatase inhibitors or selective ER degraders (SERDs).

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