Project Summary/Abstract Breast cancer is the second most prevalent type of cancer worldwide with nearly 1.7 million new cases and greater than 500,000 deaths per year. In the United States, 3 million women are living with breast cancer, representing a significant burden of disease. Of the 232,000 new cases of invasive breast carcinoma each year in the U.S., 20-25% are classified as HER2+. The advent of therapies that target HER2/ErbB2 (e.g. trastuzumab and lapatinib) has lead to improvements in the clinical management of a subset of HER2+ patients. However, adaptive resistance mechanisms of HER2+ breast cancer still hinder long-term outcomes of targeted therapies. One avenue that would improve patient outcomes is identifying and characterizing the tumor-initiating cell (TIC) for HER2+ breast cancer. Our initial studies into the TIC for HER2+ breast cancer revealed a greater tumorigenic capacity in ErbB2+ mammary stem cells (MaSCs) compared with tumors derived from luminal cells. In order to understand the variance in tumorigenic capacity, we performed transcriptomic analysis, finding a pronounced reduction of Wnt5a in tumors derived from ErbB2+ MaSCs. Further investigation revealed WNT5A secretion from luminal cells represses growth of MaSCs by a RYK- and TGFβR1-dependent phosphorylation of SMAD2. We hypothesize that WNT5A/RYK represents a paracrine-signaling module that spatiotemporally regulates the quiescence of MaSCs/TICs in ErbB2 breast cancer. To test this hypothesis, we propose the following specific aims: 1) Determine the role of lineage-specific expression of Wnt5a/Ryk in ErbB2 tumorigenesis. We will use in vivo methods for inducible Wnt5a knockout or small hairpin RNA (shRNA) targeting Ryk of preneoplastic luminal and basal cells. Our study will test the hypothesis that the inhibition of ErbB2 tumorigenesis from MaSCs depends on both luminal Wnt5a and basal Ryk expression. 2) Elucidate the molecular mechanism of WNT5A/RYK-mediated repression of MaSC growth. We will characterize the binding and signaling of WNT5A to known receptors of the mammary duct. Our study will test the hypothesis that WNT5A binds RYK preferentially leading to the phosphorylation of SMAD2. The tumor microenvironment plays integral roles in regulating cancer cell survival, metastasis, and adaptive resistance to therapy. Understanding the role of paracrine WNT5A/RYK suppression of TIC outgrowth in HER2+ breast cancer would allow for a more comprehensive knowledge of the microenvironmental interactions that regulate breast cancer initiation and progression. Elucidating the changes that must occur extrinsically (in the surrounding premalignant field) or intrinsically (in the ErbB2 TIC) which allow for the escape of WNT5A-mediated repression would improve the understanding of the paracrine regulatory network of the mammary gland and aid in improving early detection strategies and robust targeted therapies. !