Role of GPR75, the 20-hydroxyeicosatetraenoic acid (20-HETE) Receptor, in the Metastatic Features Promoted by 20-HETE in Human Castration-Resistant Prostate Cancer Cells

CARDENAS S; COLOMBERO C; AREVALOS E; COSTAS MA; NOWICKI S; PANELLO LC; FALCK J

Baltimore

Congreso; The 17th International Winter Eicosanoid Meeting; 2018

Eicosanoid Research Association, Inc.

Previous results from our group have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) availability is necessary for cell processes that contribute to metastasis in the androgen-insensitive and highly aggressive prostate cancer cell line, PC-3.Recently, the G Protein-Coupled Receptor (GPCR), GPR75 has been identified as the target for the actions of 20-HETE in the cardiovascular system. The aim of this study was to evaluate if GPR75 is involved in 20-HETE- mediated metastatic features in PC-3 cells in vitro, and to identify intracellular signaling molecules activated upon its stimulation.The expression of GPR75 protein was first confirmed in PC-3 cells and, as expected for a GPCR, its abundance was reduced by 80% by 20-HETE (100pM, 12h), and this effect was inhibited by 77% by the antagonist of the 20-HETE receptor (sodium ((6Z,15Z)-20-hydroxyeicosa-6,15-dienoyl)aspartate, AAA, 10 uM).PC-3 cells were incubated with 20-HETE (100 pM) in the presence or absence of AAA. Protein expression of Hydrogen Peroxide Inducible Clone-5 (HIC-5), E-cadherin and Vimentin (epithelial-mesenchymal transition, EMT), AKT and EGFR were assessed by western blot. The release of matrix metalloproteinase-2 (MMP-2), involved in the degradation of extracellular matrix, was assessed by zymography assay. Intracellular localization of phospho (p)- AKT and structure of actin, tubulin and vimentin were determined by immunofluorescence. Results were analyzed using one-way ANOVA followed by Dunnet?s post-hoc analysis.Incubation with 20-HETE (2h) resulted in a transient increase in the phoshorylation of EGFR, and AKT and in the translocation of p-AKT to the cell nucleus. Additionally, 20-HETE (12h) increased by 60-fold the expression of HIC-5. In every case, the effect of 20-HETE was precluded by AAA 10uM. All these cellular processes are often deregulated in malignant cells.The analysis of proteins involved in EMT showed that 20-HETE (24h) increased by 100% the expression of Vimentin (p≤0.01, n=3). This was impaired by AAA. Moreover, 20-HETE increased by 100% the release of MMP-2 into the conditioned medium (p≤0.05, n=3), and this was also inhibited by AAA. The analysis of cytoskeletal proteins distribution revealed that AAA disorganized the actin filaments throughout PC-3 cells, while the tubulin filaments remained unchanged. This suggests a role for GPR75 in the 20-HETE-prompted differentiation of PC-3 cells towards a more aggressive mesenchymal phenotype.Our results strongly suggest a role for GPR75 in 20-HETE-mediated metastatic features in PC-3 cells. However, given the pleotropic functions of 20-HETE, other intracellular pathways might be involved and their contribution to events described herein needs further analysis.