Identifying extracellular vesicle regulators in prostate cancer cells

Mbengue, Mariama 2024. Identifying extracellular vesicle regulators in prostate cancer cells. PhD Thesis, Cardiff University. Item availability restricted.

Preview	PDF - Accepted Post-Print Version Download (8MB) | Preview
	PDF (Cardiff University Electronic Publication Form) - Supplemental Material Restricted to Repository staff only Download (433kB)

Abstract

Prostate cancer (PCa) cells secrete extracellular vesicles (EVs) that exhibit many disease promoting functions. The mechanisms involved in EV generation, however, remain to be fully elucidated. We aimed to identify mechanistic factors responsible for the regulation of EV secretion in PCa cells. Candidates with a putative role in EV generation were selected based on a comprehensive database and literature interrogation. Identified candidates included Rab6a, Rab7a, Rab11b, Rab27b, Rab35, Rab37, CHMP4C, VPS28, CD81, CD9, SCAMP3, SIMPLE, Syntaxin-6, SNAP23, VAMP3, Piccolo and Synaptogyrin-2; all had some previous association with vesicle regulation but have not been comparatively tested in a single PCa model. A shRNAbased approach was successfully utilised to attenuate gene expression of most (14/17) candidates. Nanoparticle tracking analysis revealed that the target attenuations did not induce major changes in EV size nor number secreted. Immuno-affinity analysis showed that knockdown of CD9, CD81, CHMP4C and Synaptogyrin-2 had the biggest impact on vesicletetraspanin expression. RNA-sequencing analysis revealed a candidate specific impact on various transcripts with implications on stem cell-like status (CD9), RNA processing (CD81), cell division (CHMP4C) and virus processing (Synaptogyrin-2). Furthermore, most manipulations induced changes in the endolysosomal pathway, consistent with our expectation in regulation of endosomally-derived EVs. We explored the impact of modification of EV secretory processes on EV function and demonstrated that EVs from knockdown cells exhibited a reduced capacity to modulate the secretome of recipient fibroblasts. We also observed differences in knockdown EV-mediated phosphorylation of several cellular targets, indicating a knockdown dependent qualitatively distinctive fibroblast response to the EVs. The study has demonstrated some of the challenges in targeting global EV production pathways and hence this is also likely to be difficult in a therapeutic setting. Nonetheless, introducing subtle changes to the EV phenotype, rather than attenuating total EV secretion, confer downstream functional impacts that may be therapeutically exploited.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	19 August 2024
Last Modified:	20 Aug 2024 08:45
URI:	https://orca.cardiff.ac.uk/id/eprint/171461

Actions (repository staff only)

Edit Item