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Exosome Mediated Purinergic Mechanisms in Breast Cancer
AdvisorBuxton, Iain L.O.
Biochemistry and Molecular Biology
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Breast cancer is the most diagnosed cancer and the second leading cause of cancer-related deaths in women. Majority of these deaths are due to metastasis, proving that there is a great demand to understanding the mechanisms underlying metastasis. One factor released by breast cancer cells called nucleoside diphosphate kinase (NDPK) has been shown to stimulate endothelial cell proliferation, migration, and tumor-meditated angiogenesis. Extracellular NDPK generates ATP in the vicinity of the breast cancer cells to activate purinergic P2Y1 receptors on adjacent endothelial cells. The P2Y1 transactivates the VEGFR-2 on the endothelial cells to promote angiogenesis. Although NDPK was detected in conditioned media of breast cancer cells and in the serum of mice bearing tumors; the appearance of extracellular NDPK in the local tumor microenvironment has remained a mystery. We proposed that NDPK travels out of the cancer cells by small extracellular vesicles called exosomes. Exosomes were purified from breast cancer cells and characterized by transmission electron microscopy, flow cytometry, western blot analysis. These signaling vectors were also examined for angiogenic response by a tubulogenesis assay with inhibitors of NDPK and P2Y1. Further development for NDPK inhibitors were tested with a drug screen utilizing the NDPK transphosphorylation assay to identify novel small molecules that could inhibit NDPK. The in vivo role of NDPK was examined with two orthotopic mouse models while tracking the growth and development of the primary tumors and metastases. Both models were also treated with inhibitors of NDPK and P2Y1. NDPK was associated with exosomes from breast cancer cells and could stimulate tubulogenesis in endothelial cells. While ellagic acid (EA) is a known inhibitor of NDPK, several other small molecule drugs were identified as positive candidates. Treatment with EA and MRS2179, an antagonist of the P2Y1 receptor, decreased primary tumor growth and metastases in both mouse models. Exosomal NDPK supported the formation of blood vessels during tumorigenesis but may also support the growth and development of metastasis. By targeting and measuring extracellular NDPK, we could develop NDPK as a theranostic for the detection and treatment of breast cancer.