Project Summary Genome-wide sequencing of human breast tumors has revealed the remarkable molecular heterogeneity of the disease. However, the advancement of personalized breast cancer therapies requires a greater understanding of how specific genetic alterations contribute to the cellular behaviors that underlie the onset and development of breast cancer. We have recently identified a novel, transcription-independent function of the Notch1 receptor in the regulation of mammary epithelial proliferation and adherens junction organization. This proposal will utilize an interdisciplinary approach that combines a 3D tissue engineered human mammary duct platform with molecular and genetic tools to dissect cancer proliferative signaling pathways and will establish a previously undescribed, tumor suppressive Notch1 pathway in breast cancer. During the K99 phase (Aim 1), we will identify domain-specific roles of Notch1 in the transcription-independent regulation of mammary adherens junctions and cortical cytoskeleton, the signaling and proliferative pathways controlled by this non-canonical Notch1 signaling, and demonstrate the effects of transcription-independent NOTCH1 loss-of-function in breast cancer xenograft models. During the R00 phase, we will frame tumor suppressive Notch1 function in the context of mammary contact inhibition of proliferation and identify the molecular mechanisms and mechanics by which Notch1 is activated at adherens junctions during mammary tissue growth (Aim 2). In parallel, we will further leverage our biomimetic mammary duct model to explore to the distinct morphogenic phenotypes of two major recurring breast cancer mutations and test the efficacy of clinically active drugs at each stage of their tumor progression (Aim 3). The proposed research will define effects of NOTCH1 loss-of-function mutations in human breast cancer, inform therapeutic targets in patients harboring such mutations, and establish a new strategy to model breast cancer progression and assess therapies in 3D biomimetic cultures. I will gain research training in microfluidic-based, in vitro tissue engineering, as well as cancer signaling, pathology, and in vivo mouse modeling, while simultaneously enhancing career development through training in grant writing, mentoring, and leadership. I have assembled an exceptional, complementary mentoring team to help me achieve my research and career goals: Dr. Christopher Chen, expert in organotypic tissue modeling and cell mechanics, will be my primary mentor and Dr. Andrea McClatchey (MGH Cancer Center/Harvard), an international leader in cytoskeletal regulation of tumorigenesis, tumor suppressor signaling, and cancer modeling, will be my co-mentor. The institutional environment provided by the Biological Design Center at Boston University is ideally suited for this proposal and offers opportunities for scientific discussion, collaboration between biologists, clinicians, and engineers, and career development. Together, the proposed studies and career development training will ensure I achieve my goal of establishing a successful, independently-funded laboratory studying underlying mechanisms of tissue morphogenesis and tumorigenesis.