PROJECT SUMMARY The increasing number of young women diagnosed with metastatic breast cancer has awakened the urgency to define why they are more likely to suffer from aggressive breast cancers and have an increased risk of developing life-threatening brain metastasis (BM). While this has been explained by the increased frequency of aggressive triple-negative breast cancer (TNBC) in younger women, young age is a predictor of BM risk independent of tumor subtypes. Evidence has emerged from our lab that the main pre-menopausal hormone, estradiol (E2), is a key driver of BM via influencing E2-sensitive cells in the brain niche; and that endocrine therapies targeting the ER+ tumor microenvironment decrease progression of outgrowing BM only when used in combination with radiotherapy (RT) and in immunocompetent models. The scientific premise of this proposal is that E2 that endocrine therapies synergize with RT to promote effective anti-tumoral immune responses. Our long-term goal is to define how endocrine therapies and RT synergize to decrease BM to guide the translation of FDA-approved endocrine therapies to the management of TNBC BM. Our pilot data shows that endocrine therapy upregulates IRF- increases recruitment of cells to the brain. Th upregulate IRF- brain RT increases cancer-cell antigen presentation synergizing with endocrine therapy to decrease BMs (Aim 1). Our preliminary data also shows that E2 acting on ER+ astrocytes promotes upregulation of S100A4 in cancer cells, and this was blocked by endocrine therapy. While S100A4 is an extracellularly released factor that modulate myeloid cells to acquire pro-tumorigenic properties, increased expression of S100A4 has been associated with intracraneal progression and radioresistance, and high expression of S100A4 predicts radioresistance of BC cells in vitro. Thus, we propose that endocrine therapies will increase radiosensitivity of BM through blockage of E2-induced S100A4 (Aim 2). Importantly, defining how different endocrine therapies (selective-estrogen receptor modulators (SERMs), selective ER degraders (SERDs) and clinically relevant RT schedules impact anti-tumoral response and radioresistance is needed to guide future clinical trials (Aim 3). Our central hypothesis is that endocrine therapies and RT synergize by 1) upregulating IRF- astrocytes and microglia to block microglia activation and promote recruitment of T cells to the brain; and 2) by blocking S100A4 upregulation in cancer cells thus promoting radiosensitivity. Therefore, the combination of endocrine therapies with RT is essential to eliminate BMs. These studies are significant as they provide a mechanistic explanation for the increased risk of BM in young women with potential to change clinical practice. Our studies are innovative as they challenge the current dogma of how E2 contributes to tumor metastases and utilize novel transgenic models to assess the role of E2 and ERs in immune modulation in the brain.