Project Abstract/Summary Bone metastases are the major contributing factors to prostate cancer (PCa) morbidity and mortality. PCa bone metastases are uniquely osteoblastic and characterized by new bone formation, which promotes tumor growth in bone. Thus, bi-directional interaction between the PCa cells and their mis-induced bone plays a critical role in PCa progression in bone. It has been assumed that metastatic PCa induces new bone formation by stimulating the proliferation of osteoblasts in the bone marrow. However, we recently demonstrated that tumor-associated endothelial cells could give rise to osteoblasts through a little-appreciated process known as endothelial cell-to- osteoblast (EC-to-OSB) conversion, generating in the process EC-OSB hybrid cells with unique properties. Our studies showed that tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa. This new insight into the surprising bone-forming role of EC-OSB hybrid cells in PCa bone metastasis provides a novel rationale to target these cells in the tumor microenvironment. We hypothesize that tumor-induced EC-to-OSB conversion generates EC-OSB hybrid cells, which provide paracrine factors to support metastatic PCa growth in bone. Our objective is to target EC-OSB hybrid cells for improving therapy for bone metastasis. We will: Aim 1. Determine the mechanisms underlying endothelial cell-to-osteoblast (EC-to-OSB) transition. EC-to-OSB conversion requires both the inhibition of angiogenesis and activation of osteoblastogenesis pathways in endothelial cells. Our preliminary results indicate that BMP4 activates the Notch-Hey1 pathway to inhibit angiogenesis and the p38MAPK-?-catenin-OSX pathway to stimulate osteogenesis. We will examine how BMP4 integrates these two pathways to reprogram the endothelial cells. Aim 2. Identify EC-OSB hybrid cell secreted factors (EC-OSB factors) that promote PCa progression. EC-OSB hybrid cell secretome was examined by both iTRAQ (protein) and RNAseq (gene) analyses. Tenascin C was identified along with CTGF and versican, which together may constitute an ?EC-to-OSB signature?. We will investigate the effects of these EC-OSB factors on PCa cell activity in vitro and in vivo. The ?EC-OSB signature? will also be tested as biomarkers for osteoblastic bone metastasis using clinical samples. Aim 3. Develop strategies that target EC-OSB hybrid cells to improve therapy outcomes for PCa bone metastasis. The EC- OSB hybrid cells may contribute to de novo therapy resistance described previously. We will examine whether we can improve therapy outcomes for PCa bone metastasis by combining cabozantinib or cabazitaxel (targets PCa tumor) with Rad-223 (targets EC-OSB hybrid cells). Aim 4. Determine whether EC-to-OSB transition occurs in normal bone and the fraction of EC-OSB hybrid cells in tumor-induced bone. We generated a new Double Reporter Mice (col1?1-GFP/Tie2-cre/Rosa-tdTomato), in which EC are RFP+, OSB are GFP+, and EC-OSB are RFP+/GFP+, to examine whether EC-OSB hybrid cells are involved in normal bone development and their fraction in tumor-induced bone.