20-HETE/GPR75 pairing modulates the expression and transcriptional activity of the androgen receptor in androgen-sensitive prostate cancer cells.

The androgen receptor (AR) and AR-driven genes are crucial in normal and neoplastic prostate tissue. Previous results showed a link between 20-hydroxyeicosatetraenoic acid (20-HETE) production and AR-driven prostate cancer (PCa) progression. This study aims to describe the contribution of GPR75, 20-HETE membrane receptor, in 20-HETE-mediated expression and transcriptional activity of AR in PCa. In LNCaP cells, 20-HETE increased AR expression, nuclear localization, and its transcriptional activity. Also, 20-HETE enhanced dihydrotestosterone (DHT) induced effects. All was abrogated by chemical antagonism of GPR75 (19-HEDE) or its transient knockdown. In human PCa, the expression of AR-driven genes correlated with GPR75. In LNCaP xenografts, tumors from castrated animals expressed higher levels of AR, this was impaired by inhibition of 20-HETE synthesis. These data suggest that 20-HETE, through the GPR75 receptor, regulates transcriptionally active AR in PCa cells, thus making 20-HETE/GRP75 potential targets to limit the expression of AR-driven phenotype in PCa cells.

mTOR Repression in Response to Amino Acid Starvation Promotes ECM Degradation Through MT1-MMP Endocytosis Arrest.

Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.

Cytochrome 450 metabolites of arachidonic acid (20-HETE, 11,12-EET and 14,15-EET) promote pheochromocytoma cell growth and tumor associated angiogenesis.

The importance of cytochrome P450 (CYP)-derived arachidonic acid (AA) metabolites, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) as tumor growth promotors has already been described in several cancer types. The aim of this study was to evaluate the role of these compounds in the biology of pheochromocytoma/paraganglioma. These tumors originate from chromaffin cells derived from adrenal medulla (pheochromocytomas) or extra-adrenal autonomic paraganglia (paragangliomas), and they represent the most common hereditary endocrine neoplasia. According to mutations in the driver genes, these tumors are divided in two clusters: pseudo-hypoxic and kinase-signaling EETs, but not 20-HETE, exhibited a potent ability to sustain growth in a murine pheochromocytoma cell line (MPC) in vitro, EETs promoted an increase in cell proliferation and a decrease in cell apoptosis. In a mouse model of pheochromocytoma, the inhibition of CYP-mediated AA metabolism using 1-aminobenzotriazol resulted in slower tumor growth, a decreased vascularization, and a lower final volume. Also, the expression of AA-metabolizing CYP monooxygenases was detected in tumor samples from human origin, being their apparent abundance and the production of both metabolites higher in tumors from the kinase-signaling cluster. This is the first evidence of the importance of CYP- derived AA metabolites in the biology and development of pheochromocytoma/paraganglioma tumors.

GPR75 receptor mediates 20-HETE-signaling and metastatic features of androgen-insensitive prostate cancer cells.

PURPOSE: Recent studies have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is a key molecule in sustaining androgen-mediated prostate cancer cell survival. Thus, the aim of this study was to determine whether 20-HETE can affect the metastatic potential of androgen-insensitive prostate cancer cells, and the implication of the newly described 20-HETE receptor, GPR75, in mediating these effects. METHODS: The expression of GPR75, protein phosphorylation, actin polymerization and protein distribution were assessed by western blot and/or fluorescence microscopy. Additionally, in vitro assays including epithelial-mesenchymal transition (EMT), metalloproteinase-2 (MMP-2) activity, scratch wound healing, transwell invasion and soft agar colony formation were used to evaluate the effects of 20-HETE agonists/antagonists or GPR75 gene silencing on the aggressive features of PC-3 cells. RESULTS: 20-HETE (0.1 nM) promoted the acquisition of a mesenchymal phenotype by increasing EMT, the release of MMP-2, cell migration and invasion, actin stress fiber formation and anchorage-independent growth. Also, 20-HETE augmented the expression of HIC-5, the phosphorylation of EGFR, NF-κB, AKT and p-38 and the intracellular redistribution of p-AKT and PKCα. These effects were impaired by GPR75 antagonism and/or silencing. Accordingly, the inhibition of 20-HETE formation with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) elicited the opposite effects. CONCLUSIONS: The present results show for the first time the involvement of the 20-HETE-GPR75 receptor in the activation of intracellular signaling known to be stimulated in cell malignant transformations leading to the differentiation of PC-3 cells towards a more aggressive phenotype. Targeting the 20-HETE/GPR75 pathway is a promising and novel approach to interfere with prostate tumor cell malignant progression.

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10 µM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 µM, 23 ± 3%*; 10 µM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 µM, 16 ± 4%*; 10 µM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*-42%* by 1-10 µM HET0016. Incubation with 20-HETE (5-1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 µM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.

Defective renal dopamine function and sodium-sensitive hypertension in adult ovariectomized Wistar rats: role of the cytochrome P-450 pathway.

We have previously shown that ovariectomy in adult Wistar rats under normal sodium (NS) intake results in an overexpression of the total Na(+)-K(+)-ATPase (NKA) α1-subunit (Di Ciano LA, Azurmendi PJ, Toledo JE, Oddo EM, Zotta E, Ochoa F, Arrizurieta EE, Ibarra FR. Clin Exp Hypertens 35: 475-483, 2013). Upon high sodium (HS) intake, ovariectomized (oVx) rats developed defective NKA phosphorylation, a decrease in sodium excretion, and an increment in mean blood pressure (MBP). Since NKA phosphorylation is modulated by dopamine (DA), the aim of this study was to compare the intracellular response of the renal DA system leading to NKA phosphorylation upon sodium challenge in intact female (IF) and oVx rats. In IF rats, HS caused an increase in urinary DA and sodium, in NKA phosphorylation state, in cytochrome P-4504A (CYP4A) expression, and in 20-HETE production, while MBP kept normal. Blockade of the D1 receptor (D1R) with the D1-like receptor antagonist SCH 23390 in IFHS rats shifted NKA into a more dephosphorylated state, decreased sodium excretion by 50%, and increased MBP. In oVxNS rats, D1R expression was reduced and D3R expression was increased, and under HS intake sodium excretion was lower and MBP higher than in IFHS rats (both P < 0.05), NKA was more dephosphorylated than in IFHS, and CYP4A expression or 20-HETE production did not change. Blockade of D1R in oVxHS rats changed neither NKA phosphorylation state nor sodium excretion or MBP. D2R and PKCα expression did not vary among groups. The alteration of the renal DA system produced by ovariectomy could account for the defective NKA phosphorylation, the inefficient excretion of sodium load, and the development of salt-sensitive hypertension.

Role of 3 lipoprotein lipase variants in triglycerides in children receiving highly active antiretroviral therapy.

BACKGROUND: Lipoprotein lipase is a key enzyme in lipid metabolism, especially for plasma triglycerides (TGs). Genetic variants have been associated with lipid levels in healthy individuals, cardiovascular disease, obesity and diabetes. Our aim was to evaluate the influence of 3 polymorphisms: Hind III, Pvu II and S447X in plasma TG levels in human immunodeficiency virus-1-infected children under highly active antiretroviral therapy (HAART). METHODS: Fifty-two children diagnosed with human immunodeficiency virus-1 between 2005 and 2009 were retrospectively selected with at least 1 plasma TG level assessment. TG levels were examined before and after 1 year of HAART. Hypertriglyceridemia was defined as TG > 150 mg/dL. Hind III (H+/H-), Pvu II (P+/P-) and S447X (S/X) were determined by polymerase chain reaction and restricted fragment length polymorphism. The Wilcoxon sum-rank test was used to compare median plasma TG among groups. Also, allelic frequencies were estimated for these variants in an Argentinean population. RESULTS: Allelic frequencies for human immunodeficiency virus-1-infected children were: H-, 0.21; P-, 0.53; and X, 0.05 with no significant differences to controls. After 1 year of HAART, median TG levels were significantly lower in P-/P- (98.5 mg/dL) when compared with P+/P+ (180 mg/dL) (P = 0.039). The presence of the P- allele was associated with an 11-fold lower risk of hypertriglyceridemia. Hind III and S447X were not associated with TG at the selected time points. CONCLUSIONS: Our findings suggest a protective effect of lipoprotein lipase polymorphisms against hypertriglyceridemia in children after 1 year of HAART. These results could endorse a prompt nutritional or pharmacological intervention in patients lacking the P- allele.

Cytochrome P4504A inhibitors attenuate the exaggerated natriuretic response to volume expansion in thyroidectomized rats.

Thyroidectomy augments the natriuretic response to volume expansion; however, the mechanism remains unknown. This study assessed the role of 20-hydroxyeicosatetraenoic acid (20-HETE) in the natriuretic response to an acute volume expansion in hypothyroid rats. Urine flow (1.9-fold), sodium excretion (2.4-fold), fractional sodium excretion (3.8-fold), and distal delivery of sodium (4.1-fold) increased to a greater extent in thyroidectomized rats (TX) than in sham-operated controls (SHAM) following i.v. infusion of isotonic saline (5% body weight) over 60 min. This was associated with inhibition of both proximal and distal tubular reabsorption of sodium. Administration of two mechanistic and chemical dissimilar inhibitors of the synthesis of 20-HETE, 1-aminobenzotriazole (ABT), and N-hydroxy-N'-(-4-butyl-2-methylphenyl)formamidine (HET0016) decreased the natriuretic response in TX rats. Glomerular filtration rate was lower in TX than in SHAM rats and was not altered by the CYP4A inhibitors. The expression, intrarenal distribution, and the formation of 20-HETE and expoxygenase metabolites of arachidonic acid were similar in the cortex and medulla of SHAM and TX rats. These results suggest that CYP4A-derived metabolites of arachidonic acid play an important role in the enhanced natriuretic response to volume expansion in hypothyroid rats even though TX did not alter the expression or activity of these enzymes.