The studies outlined in this proposal focus on the mechanisms by which aggressive breast cancer cells generate large numbers of exosomes with unique cargo, together with a total secretome that significantly enhances their potential for metastatic spread. They are based on exciting developments in the cancer biology field which show that exosomes, a major class of extracellular vesicles (EVs), play important roles in a number of aspects of cancer progression. These include the ability of exosomes to confer tumor cells with the capability to show resistance to chemotherapeutic reagents as well as to immune therapy, together with their roles in promoting metastatic spread. We recently discovered that the downregulation of SIRT1 by aggressive breast cancer cells has an important influence on the numbers of exosomes that they generate, the nature of the exosome cargo, as well as the composition of their total secretome. This is due to the NAD+-dependent deacetylase/deacylase Sirtuin (SIRT1) playing a key role in maintaining normal lysosomal function through a novel mechanism that ensures the proper expression of a major subunit of the vacuolar ATPae (v-ATPase). We also have recently found that the formation and shedding of exosomes appear to be dependent on the elevations in glutamine metabolism characteristic of breast cancer cells (i.e. their ‘glutamine addiction’). These findings now raise important questions regarding how the dependence of aggressive breast cancer cells on glutamine metabolism influences and/or works together with the down-regulation of SIRT1 expression/activation to regulate lysosomal function and exosome biogenesis, thus producing a secretome that stimulates cancer cell invasiveness and helps drive the metastatic process. The different laboratories participating in this proposal will take advantage of their multi-disciplinary expertise in biochemical and chemical biology approaches in probing cancer cell metabolism and exosome biogenesis, high-resolution imaging, 3D spheroid culture and tumor organoids, and the use of mouse models, in probing three key aspects of the mechanisms driving breast cancer metastasis. These are: 1) Examining the relationship between SIRT1 down-regulation, elevated glutamine metabolism and the generation exosomes with unique cargo by aggressive breast cancer cells. 2) Understanding how SIRT1 down-regulation impacts vacuolar ATPase expression to generate a secretome capable of promoting cancer cell invasiveness. 3) Determining how SIRT1 expression/activity affects exosome production, cell invasiveness and metastatic spread in breast cancer models. The expectation is that these studies will lead to the identification of exciting new treatment strategies for the devastating effects of aggressive breats cancers, and ultimately, for other metastatic diseases.