CEDIE   05498
CENTRO DE INVESTIGACIONES ENDOCRINOLOGICAS "DR. CESAR BERGADA"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
GPR75, The 20-hydroxyeicosatetraenoic Acid (20-HETE) Receptor Has A Role In The Metastatic Features Promoted By 20-HETE In Human Castration- Resistant Prostate Cancer Cells
Autor/es:
NOWICKI S.; ARÉVALOS E.; COLOMBERO C.; PANELO L.; COSTAS M.; CÁRDENAS ALCOSER ELENA SOFÍA; FALCK J.R.; NOWICKI S.; ARÉVALOS E.; COLOMBERO C.; PANELO L.; COSTAS M.; CÁRDENAS ALCOSER ELENA SOFÍA; FALCK J.R.
Lugar:
Chicago
Reunión:
Congreso; 100th Annual Meeting & Expo of The Endocrine Society (ENDO); 2018
Institución organizadora:
The Endocrine Society
Resumen:
20-hydroxyeicosatetraenoic acid (20-HETE), the product from the 20- hydroxylation of arachidonic acid by cytochrome P450 isoforms (CYP4F2 and CYP4A11), has been implicated in the oncogenesis of several types of tumors. Previous results from our group have shown that 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 in vitro if GPR75 is involved in 20-HETE- mediated metastatic features in PC-3 cells, and to identify intracellular signaling molecules activated upon its stimulation.PC-3 cells were incubated with 20-HETE (100 pM) in the presence or absence of an antagonist of the 20-HETE receptor (sodium ((6Z,15Z)-20-hydroxyeicosa-6,15-dienoyl)aspartate, AAA, 10 uM). Protein expression of GPR75, 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.The expression of GPR75 was first confirmed in PC-3 cells and, as expected for a GPCR, its abundance was reduced by 80% by 20-HETE (12h), and this effect was inhibited by AAA by 77%.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.