Investigating the role of TRPC3 in the biogenesis of extracellular vesicles
Cells within multicellular organisms transcribe, package and release nano-sized spheres called extracellular vesicles (EVs) that carry an array of biomolecules that can be transferred between cells. EV-mediated communication contributes to a wide range of physiological and pathological processes including cancer progression, yet the molecular mechanisms underlying their formation and release remain incompletely characterised. Elevated intracellular calcium (Ca2+) induces EV release in a variety of cell types - with preliminary data to suggest the Ca2+ permeable transient receptor potential (TRP) channel TRPC3 may facilitate Ca2+-mediated EV biogenesis. TRPC3 contributes to the progression of ovarian cancer (OC) in vitro and in vivo, but its role in EV biogenesis and how this influences the pathogenesis of OC is unknown. This thesis therefore aimed to investigate the role of TRPC3 and TRPC3-mediated EV communication in ovarian cells, and how TRPC3 activation, Ca2+ influx and upstream GPCR stimulation influence EV biogenesis and cargo. Initial experiments detected TRPC3 expression across a panel of ovarian cell lines, with SKOV3 chosen as the principal line of study. Activating TRPC3 amplified Ca2+ signalling in SKOV3 cells, and significantly increased cell growth and migration, whilst cell invasion was unchanged. In contrast, inhibiting TRPC3 attenuated histamine-induced Ca2+ signalling, indicating that TRPC3 is activated downstream of histamine GPCRs. To enable specific detection and analysis of SKOV3-EVs, EVs were extracted in serum-free media which yielded a purer population of EVs that present typical markers and morphology. TRPC3 activation induced the release of EVs that promoted recipient SKOV3 cell growth, but bore no effect on SKOV3 motility or invasiveness. Preliminary results also indicated that TRPC3 and its upstream regulator mGluR1 are incorporated into SKOV3-EVs. Using live single-cell imaging, histamine, Ca2+, and TRPC3 activation were shown to stimulate the release of EVs with distinct characteristics and fusion dynamics, including localised, synchronised EV release. Proteomic profiling of these treatment- induced EVs revealed previously unappreciated roles for CD81, NDPKs and Ca2+-dependent interactions with the syndecan-syntenin-ALIX axis in EV biogenesis. Proteomic analysis also evidenced the endosomal origin and enriched metastatic potential of TRPC3 activator-induced EVs, as well as presenting novel characterisation of the effect of DMSO on EV proteomes. Overall, these results present strong evidence that TRPC3 mediates EV biogenesis in ovarian cells, and through altering EV characteristics and proteomic cargo, contributes to the development of OC. This thesis provides a novel contribution to our understanding of the molecular mechanisms and dynamics which underly EV biogenesis, with potential therapeutic applications in cancer.
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