Oncogenic Extracellular Vesicle Biogenesis and Protein Trafficking

Extracellular vesicles (EVs) include exosomes and microvesicles, and are important mediators of cell-to-cell communication in healthy and pathological environments. Packed with biological information and present in a variety of biological fluids, EVs offer exciting promise for biomarker discovery and applications in therapeutics and non-invasive diagnostics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis, cargo packaging, and secretion remain unknown. To an extent, these findings have been restricted by the limited cells from which vesicles have been characterized utilizing the same enrichment method. The following work contributes to advancement of knowledge surrounding EV formation within the cell, protein trafficking into EVs, and the content and functions of oncogenic vesicle secretion in cancer. In Chapter 2 of this dissertation, vesicle secretion is characterized across sixty cancer cell lines from the National Cancer Institute (NCI-60) by nanoparticle tracking analysis. The quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Positive correlates of exosomal-sized vesicle secretion included Rab GTPases and mediators of sphingolipid metabolism, while larger microvesicle-sized vesicle secretion was associated with gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. To investigate the global protein content of NCI-60 cell derived EVs, pure vesicle isolates were harvested for a large-scale mass spectrometry approach, detailed in Chapter 3. This work provides the largest proteomic profile of EVs in a single study, identifying 6,071 proteins with 213 common to all isolates. Proteins included established EV markers, and vesicular trafficking proteins such as Rab GTPases and tetraspanins. The identification of differentially-expressed proteins may offer future candidates for earlier cancer diagnosis and prognostic monitoring of disease. Network analysis of vesicle quantity and proteomes identified EV components associated with vesicle secretion, including CD63, CD81, syntenin-1, VAMP3, Rab GTPases, and integrins. Finally, integration of vesicle proteomes with whole-cell molecular profiles revealed similarities, suggesting EVs provide a reliable reflection of their progenitor cell content, and are therefore excellent indicators of disease. Together, these observations reveal new insights into key players involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. In both the aforementioned works, a tetraspanin protein, CD63 was identified and confirmed for its role in small vesicle biogenesis. CD63 is a conserved protein enriched in late endosomal and lysosomal compartments and has been widely used as an exosome marker. However, little is understood about the mechanisms of CD63-mediated exosome formation, or its interacting proteins. Accumulating evidence has suggested that an Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1) may interact with CD63 within the exosomal pathway. Viruses share similar sizes and structures as exosomes, and recent evidence suggests that viruses can hijack the exosome pathway to modulate cell-to-cell signaling. Indeed, LMP1 trafficking into multivesicular bodies (MVBs) can alter the content and function of exosomes. LMP1-modified exosomes can in turn enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. In efforts to advance knowledge surrounding the role of CD63 in exosome production and vesicular protein sorting, original work in Chapter 4 demonstrates the importance of CD63 presence for exosomal packaging of LMP1. In this study, a role of LMP1 in vesicle production that requires CD63 is described, providing an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Further evidence revealed the role of CD63 in limiting LMP1-induced non-canonical NF-κB and ERK activation. Overall, these findings have implications in future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in the context of viral- or nonviral- associated tumorigenesis.