PROJECT SUMMARY The tissue microenvironment dictates adult stem cell behavior during normal homeostasis and cancer. The microenvironment of the breast contains numerous distinct cell types including basal and luminal mammary epithelial cells that form a ductal network surrounded by stromal cell types such as adipocytes and fibroblasts. One of the key regulators of the tumor microenvironment are the matrix metalloproteinases (MMPs), which are secreted proteolytic enzymes that affect multiple aspects of cancer including extracellular matrix remodeling, inflammation, and metastasis. MMPs have been implicated in breast cancer for many years and they have been considered as therapeutic targets to treat cancer patients. However, previous clinical cancer trials using synthetic small molecule inhibitors to interfere with MMP catalytic activity yielded disappointing results. Our previous research has shown that MMPs such as MMP3 may work in a non-proteolytic manner through their hemopexin domain. This may explain why compound inhibitors targeting the catalytic function of MMPs failed to improve disease outcome. Here, we will focus on the role of stromal fibroblast-derived MMP3 and its interaction with the non- canonical Wnt signaling pathway during BRCA1-related breast cancer initiation. In our previous work, we have established that MMP3 can modulate the non-canonical Wnt signaling pathway by binding and inactivating Wnt5b via the hemopexin domain and that this plays an important role in stem cell maintenance during normal homeostasis of the mammary epithelium. We will now determine whether this interaction of MMP3 with Wnt5b affects breast cancer initiation. In particular we will employ lentiviral transduction, transplantation of mammary epithelial cells, mouse models of breast cancer in combination with cutting single cell genomics and multi- modal imaging tools to address whether (1) fibroblast-derived MMP3 promotes pro-tumorigenic changes in breast epithelial cells in vitro and in vivo in a potentially non-proteolytic manner, (2) Wnt5b is the critical substrate mediating the tumor-promoting effects associated with MMP3, (3) Wnt5b specifically regulates mammary stem cell proliferation and exerts tumor suppressing effects within the breast epithelium. Taken together, these studies will unravel new molecular mechanisms through which the microenvironment promotes tumor initiation, and yield a better understanding of the non-canonical Wnt signaling pathway in regulating normal epithelial homeostasis and breast cancer initiation. Ultimately, our work may implicate the MMP3/Wnt5b axis as a potential therapeutic target for primary cancer prevention in BRCA1 mutation carriers.