Imaging the interactions and inhibition of human group IIA phospholipase A₂ in prostate cancer cells

Abstract

Dysregulation of eicosanoid production plays a crucial role in inflammation-driven prostate cancer (PCa). The inflammatory protein secreted phospholipase A2 (hGIIA), is aberrantly expressed in PCa, causes overproduction of eicosanoids and subsequent proliferation, making hGIIA an attractive therapeutic target. hGIIA’s role as an amplifier of inflammation extends past eicosanoid production and is likely to be due, at least in part, to a catalytically independent mechanism, involving the activation of cytosolic phospholipase A2 alpha (cPLA2-α). This thesis aims to characterise this mechanism, in which the cytoskeletal protein vimentin and epidermal growth factor receptor (EGFR) have been implicated. Furthermore, the mechanism of action of c2, a cyclic pentapeptide derived from hGIIA with known anti-cancer properties, was also explored.

Chapter three investigated the interaction between hGIIA and vimentin. Endogenous hGIIA, present within caveolae, colocalised with vimentin in PCa cell lines PC-3 and LNCaP, but not DU145.

Chapter four examined the pathway of hGIIA entry into PCa cells and whether it can initiate calcium signalling and prostaglandin E2 (PGE2) production.

Chapter five explored the mechanism of action of hGIIA inhibitor, c2. Using an in vitro enzyme immunoassay, c2 was identified to block the interaction of hGIIA with coil 2 of vimentin more potently than any tested clinically used compound with potential vimentin binding properties.

A new model was therefore proposed that hGIIA stimulates eicosanoid production and proliferation through previously reported binding to EGFR, triggering cPLA2-α activation. As c2 is able to reduce both eicosanoid production and hGIIA trafficking, this indicates its bifunctional ability to block hGIIA activation of EGFR, as well as its observed inhibition of the hGIIA/vimentin interaction.

The findings from this thesis highlight that hGIIA contributes to PCa pathology via a catalytically independent mechanism and elucidates it as an attractive therapeutic target. c2 has been identified as a potent inhibitor of this mechanism and though unproven, its potential clinical uses could extend past PCa to other inflammation driven diseases with overexpressed hGIIA.

Date of Thesis submission	2022
Original language	English
Awarding Institution	Western Sydney University
Supervisor	Kieran Scott (Supervisor), Anya Salih (Supervisor) & Enrico Gratton (Supervisor)