PROJECT SUMMARY Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene best known for is function in DNA repair. Early studies suggested that genome instability within the mammary epithelial system of BRCA1 mutation carriers (BRCA1+/mut) promotes a cascade of cell autonomous, genetic events ultimately giving rise to cancer initiation within a population of luminal epithelial progenitor cells. However, a major gap in knowledge is whether germline BRCA1 mutations lead to cell state aberrations within the cellular microenvironment surrounding the epithelium, which may act in trans to promote pre-cancerous changes in the epithelial system thereby promoting breast cancer initiation. In this renewal, we will expand on the provocative findings from our currently funded project, which identified dramatic precancerous changes in both breast epithelium and surrounding microenvironment of human BRCA1+/mut. Our work identified the expansion of epithelial cells with basal-luminal intermediate (BLI) phenotype as well as a population of epithelium-surrounding precancer-associated fibroblasts (preCAFs) expressing copious amounts of matrix metalloproteinase 3 (MMP3) and immunoregulatory factors (e.g., neutrophil-attracting CXCL8). Based on our progress and preliminary data, we will address the hypothesis that preCAFs emerge in a cell-intrinsic manner due to fibroblast-specific BRCA1 deficiency leading to an aberrant form of fibroblast differentiation. In this context, we will test whether cell-intrinsic NFkB activation in BRCA1+/mut acts as a central signaling node towards the preCAF state. We will also determine whether BRCA1mut fibroblasts establish a precancerous immunosuppressive microenvironment during breast cancer initiation through MIP-2, a mouse homologue for CXCL8. Addressing these major gaps in knowledge about the function and origin of preCAFs may pave the way towards novel therapeutic approaches to block the cancer-promoting function of preCAFs or reverse their phenotype as precision cancer prevention strategies.