20-Hydroxyeicosatetraenoic acid (20-hete) promotes a malignant phenotype in human castration-resistant prostate cancer cells through stimulation of the g protein-coupled receptor gpr75

20-Hydroxyeicosatetraenoic acid (20- HETE), the product of 20-hydroxylation of arachidonic acid by cytochrome P450 isoforms (CYP4F2 and CYP4A11), has a role in the oncogenesis of several human tumors. Recently, the GPR75 receptor has been identified as the target for 20-HETE. We have shown that androgen independent prostate cancer cells (PC-3) express GPR75. The aim of this study was to assess in vitro if 20-HETE/GPR75 modify the metastatic features of PC-3 cells. Cells were incubated with 20-HETE or its stable analog 5,14- HEDGE (both 0.1 nM) in the presence or absence of two different antagonists of the 20-HETE receptor, AAA or 19-HEDE (both 5 or 10 uM). The following assays were performed: e-cadherin and vimentin protein expression (epithelial-mesenchymal transition), zymography (release of matrix metalloproteinase-2 (MMP-2)), immunofluorescence and p-FAK (changes of cytoskeleton), scratch wound healing (migration), and soft agar colony formation (anchorage-independent growth). Results were analyzed using one-way ANOVA followed by Dunnet’s. 20-HETE (24 h) increased by 150 % the expression of vimentin (p<0.0001, n= 3) and diminished by 40 % the expression of ecadherin (p<0.0001, n= 3), whereas these effects were reversed by AAA (p<0.0001 and p<0.05, respectively). 20-HETE increased by 52 % the release of MMP-2 (p<0.05, n= 3), and this was also inhibited by AAA (p<0.001). AAA disorganized the actin filaments throughout PC-3 cells, while tubulin filaments remained unchanged. Also, 20-HETE increased by 89 % FAK phosphorylation (Y397) (p<0.0001, n= 3). 20-HETE increased by 147 % cell migration rate (p<0.0001, n= 3) and this effect was reverted by both antagonists, AAA or 19-HEDE (p<0.05 and p<0.0001, respectively), or by knockdown of GPR75 (p<0.0001). Finally, 5,14-HEDGE (21 days) formed twice the number of colonies vs. control (p<0.05, n= 2) and this was abolished by AAA (p<0.05). These results strongly suggest a role for GPR75 in 20-HETE-mediated metastatic features in PC-3 cells. Fil: Cardenas, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Colombero, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina