PROJECT SUMMARY Breast cancer is a widespread disease that will claim the lives of over 40,000 US women in 2022 alone, with the majority of these deaths resulting from stage IV disease, in which cancer has metastasized to distant regions, as metastatic disease is difficult to treat and has few effective treatment options. Breast cancer cells metastasize by invading the basement membrane, invading into blood vessels, circulating to distant tissues, leaving the blood vessel, and colonizing that tissue. The epithelial cell adhesion molecule (EpCAM) plays a role in cell adhesion, migration, and invasion, and has been linked to several epithelial cancers, including breast, prostate, and colorectal cancer. Despite its potential to facilitate several steps of the metastatic cascade and its link to epithelial cancers, the specific role of EpCAM in metastasis remains unknown. In his study of normal tissues, primary tumors, and metastatic nodules of 17 metastatic breast cancer patients, our collaborator Dr. Pedram Argani determined that EpCAM protein expression was increased in metastatic nodules, without a corresponding increase in EpCAM mRNA expression. We therefore hypothesize that post-translational modification of EpCAM, which has three known N-glycosylation sites and no known O-glycosylation sites, stabilizes and increases the abundance of EpCAM in metastatic tissues. Our recent study of these same samples found that the abundance of N-glycans was significantly increased in metastatic tissues versus primary tumors versus normal tissues and identified 25 significantly differentially abundant N-glycans of interest. Our study also found that EpCAM protein expression was significantly increased in metastatic tissues compared to primary tumors and was significantly statistically correlated with the expression of seven N-glycans. Taken together, these data suggest that EpCAM N-glycosylation may play a role in metastatic progression. The central hypothesis of this project is that increases and changes in N-glycosylation stabilize EpCAM and allow it to facilitate metastasis. I propose to test this hypothesis through the following specific aims: Aim 1: To elucidate the impact of EpCAM N-glycosylation on EpCAM stability and adhesion in human breast cancer cells; Aim 2: To assess the impact of EpCAM N- glycosylation on metastasis in mouse models of breast cancer. These aims will be achieved through a combination of cancer biology, biochemistry, and mass spectrometry approaches in human triple-negative breast cancer cell lines and breast cancer xenograft models. This work is significant because it will elucidate the role of N-glycosylation of EpCAM in metastatic breast cancer, determine the specific N-glycosylation profile of EpCAM in metastasizing breast cancer cells, and may subsequently uncover novel therapeutic targets and strategies in metastatic breast cancer.