Summary Brain metastasis accounts for significant morbidity and mortality of many cancer types, the two most common being lung and breast cancers. This devastating complication is initiated by a rare subset of the circulating tumor cells (CTCs) shed into the blood stream. The crossing of the blood-brain barrier (BBB) by CTCs is the first critical step that initiates brain metastasis. Thus, understanding how CTCs can breach the BBB is critical for developing means to predict, prevent, or treat brain metastasis. Our recent study using ex vivo expanded CTCs isolated from breast cancer patients identified a transmembrane surface receptor, SEMA4D, promotes brain metastasis by enabling CTCs to cross the BBB via binding to its ligand PLXNB1 on the brain endothelial cells (BECs). Our preliminary data now show that SEMA4D stimulation leads to YAP/TAZ signaling in tumor cells, a pathway well known to promote cell motility and migration, and recently implicated in the development of brain metastasis. Therefore, we hypothesize a role for “reverse signaling” through the SEMA4D cytoplasmic tails to activate YAP signaling for synergizing with SEMA4D-PLXNB1 tethering to the BECs to breach the BBB. We will utilize our unique resources of patient-derived CTCs, and in vitro and in vivo BBB transmigration assays to test our hypothesis. The overall objective of this proposal is to dissect the detailed mechanisms linking SEMA4D to YAP signaling, and the role for YAP in SEMA4D-mediated BBB transmigration. Delineating the combinatory effect of SEMA4D and YAP signaling will allow us to design potential therapeutic approaches to prevent SEMA4D-mediated brain metastasis. Such knowledge will also provide a mechanistic basis for identifying additional BBB transmigration initiators that could act similarly through receptor tethering to BECs and subsequent activation of YAP, which could have a broader impact on identifying predictive biomarkers for brain metastasis.