Regulation and function of exosomal cargo loading in cancer progression Exosomes are 50-150 nm, endosome-derived, extracellular vesicles that are secreted by most cells. The exosomes secreted by tumor cells contain increased levels of proteins with oncogenic and immunosuppressive functions and play important roles in tumor progression, immunosuppression and therapeutic resistance. Exosomal cargo loading is a highly regulated process, and ESCRTs (Endosomal Sorting Complex Required for Transport) are believed to be critically involved in this process. ESCRTs function by promoting membrane invagination and vesicle formation, recognizing and linking cargoes to the membrane and facilitating the release of the vesicles containing the cargoes into the lumen of endosomes. Although multiple ESCRT components have been reported to have oncogenic or tumor suppressor function, systematic studies of the roles of ESCRT components in tumor-derived exosomes are still lacking. My long-term research goal is to understand the regulation of exosomal cargo loading and the contribution of specific exosomal sorting to tumorigenesis. HD-PTP (His Domain Protein Tyrosine Phosphatase) is an ESCRT associated adaptor protein with putative phosphatase activity encoded by the PTPN23 gene, located on human chromosome 3p21.3, which is frequently deleted in tumors. As a tumor suppressor, frequent PTPN23 hemizygous deletions, mutations, and reduced HD-PTP expression have been detected in cancer patients. However, the mechanisms for tumor suppressor function of HD-PTP are poorly understood. My preliminary studies show that loss of HD- PTP significantly increases exosomal PD-L1 levels in triple negative breast cancer (TNBC) cells and suggest that the tumor promoting effects of HD-PTP loss is mediated by exosomal PD-L1. Thus, I hypothesize that HD- PTP functions as a tumor suppressor, loss of which promotes tumor progression through increasing exosomal PD-L1 level.To test the hypothesis, I will first examine whether the effect of HD-PTP on exosomal PD-L1 is mediated by its ESCRT function or phosphatase activity or both. I will also further investigate whether increased exosomal PD-L1 level induces the tumor promoting effect of HD-PTP loss that I observe in mouse models. Next, I will study the mechanisms for HD-PTP-dependent regulation of exosomal and cellular PD-L1 expression. For this purpose, I will first examine the effects HD-PTP loss on localization, intracellular trafficking and posttranslational modifications of PD-L1. CRISPR/Cas9 screen will be performed to look for novel pathway that mediates the effect of HD-PTP on cell surface PD-L1. Finally, I will perform exosomal proteomics studies to systematically explore the effects of dysregulation of ESCRT machinery on exosomal content change and the exosome-mediated function in tumor progression, immune response and therapeutic resistance. In summary, this research will unveil the novel functions of ESCRTs in tumor progression, ena...