Convergence of eicosanoid and integrin biology: Role of Src in 12-LOX activation.

12-Lipoxygenase (12-LOX) metabolizes arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, or 12(S)-HETE, a proinflammatory bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. The mechanisms underlying 12-LOX-mediated signaling in cancer progression are still ill-defined. In the present study we demonstrate that 12-LOX phosphorylation and subsequent enzymatic activity occurs after integrin ß4 stimulation and Src kinase recruitment to the integrin subunit. Inhibition of Src activity by PP2 or Src dominant-negative mutants reduced 12-LOX tyrosine phosphorylation and 12(S)-HETE production in response to integrin ß4 stimulation in A431 cells. The pertinent Src-targeted residues for 12-LOX activity were mapped to Y19 and Y614, where 12-LOX mutants Y19F and Y614F showed 70% less enzymatic activity. Furthermore, we have shown that the 12-LOX activity modulated by these residues impacts migration. To our knowledge, this is the first report that c-Src kinase activity is required for ß4-integrin-mediated phosphorylation of 12-LOX.

12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Previously we identified and deorphaned G-protein-coupled receptor 31 (GPR31) as the high-affinity 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] receptor (12-HETER1). Here we have determined its distribution in prostate cancer tissue and its role in prostate tumorigenesis using in vitro and in vivo assays. Data-mining studies strongly suggest that 12-HETER1 expression positively correlates with the aggressiveness and progression of prostate tumors. This was corroborated with real-time PCR analysis of human prostate tumor tissue arrays that revealed the expression of 12-HETER1 positively correlates with the clinical stages of prostate cancers and Gleason scores. Immunohistochemistry analysis also proved that the expression of 12-HETER1 is positively correlated with the grades of prostate cancer. Knockdown of 12-HETER1 in prostate cancer cells markedly reduced colony formation and inhibited tumor growth in animals. To discover the regulatory factors, 5 candidate 12-HETER1 promoter cis elements were assayed as luciferase reporter fusions in Chinese hamster ovary (CHO) cells, where the putative cis element required for gene regulation was mapped 2 kb upstream of the 12-HETER1 transcriptional start site. The data implicate 12-HETER1 in a critical new role in the regulation of prostate cancer progression and offer a novel alternative target for therapeutic intervention.-Honn, K. V., Guo, Y., Cai, Y., Lee, M.-J., Dyson, G., Zhang, W., Tucker, S. C. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Platelet-type 12-lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF-κB.

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12-lipoxygenase (12-LOX) occurred more frequently in advanced stage, high-grade prostate cancer, suggesting that 12-LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12-LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12-LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12-LOX in prostate cancer PC-3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)-hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12-LOX, is able to increase MMP9 expression in wild-type PC-3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12-LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor-kappa B (NF-κB)-driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12-LOX, PI3K or NF-κB inhibited MMP9 expression in 12-LOX-expressing PC-3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12-LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF-κB signaling. The role of 12-LOX-mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12-LOX in prostate cancer.

Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid.

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[(3)H]HETE (K(d) = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC(50) = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.

Thromboxane A2 receptors in prostate carcinoma: expression and its role in regulating cell motility via small GTPase Rho.

Thromboxane A(2) (TxA(2)) is a prostanoid formed by thromboxane synthase using the cyclooxygenase product prostaglandin H(2) as the substrate. Previously, increased expression of thromboxane synthase was found in prostate tumors, and tumor cell motility was attenuated by inhibitors of thromboxane synthase. This study was undertaken to elucidate how tumor motility is regulated by TxA(2). Here, we report that human prostate cancer cells express functional receptors for TxA(2) (TP). Ligand binding assay found that PC-3 cells binded to SQ29548, a high-affinity TP antagonist, in a saturable manner with K(d) of 3.64 nmol/L and B(max) of 120.4 fmol per million cells. Treatment of PC-3 cells by U46619, a TP agonist, induced PC-3 cell contraction, which was blocked by pretreatment with the TP antagonist SQ29548 or pinane TxA(2). The migration of prostate cancer cells was significantly inhibited either by sustained activation of TP or by blockade of TP activation, suggesting that TP activation must be tightly controlled during cell migration. Further studies found that small GTPase RhoA was activated by TP activation, and pretreatment of PC-3 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. A dominant-negative mutant of RhoA also blocked U46619-induced cell contraction. Taken together, the data suggest that TPs are expressed in prostate cancer and activation of TPs regulates prostate cancer cell motility and cytoskeleton reorganization through activation of Rho.

Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells.

12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility.

Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens.

This report describes Pi transport activity in chondrocytes isolated from the growth plate (GP) of normal adolescent chickens grown in primary cell culture. Our recent work showed that Pi transport in matrix vesicles (MV) isolated from normal GP cartilage was not strictly Na+-dependent, whereas previously characterized Pi transport from rachitic GP cartilage MV was. This Na+-dependent Pi transporter (NaPiT), a member of the Type III Glvr-1 gene family, is expressed only transiently during early differentiation of GP cartilage, is enhanced by Pi-deficiency, and is most active at pH 6.8. Since GP mineralization requires abundant Pi and occurs under slightly alkaline conditions, it seemed unlikely that this type of Pi transporter was solely responsible for Pi uptake during normal GP development. Therefore we asked whether the lack of strict Na+-dependency in Pi transport seen in normal MV was also evident in normal GP chondrocytes. In fact, cellular Pi transport was found not to be strictly Na+-dependent, except for a brief period early in the culture. Choline could equally serve as a Na+ substitute. Activity of choline-supported Pi transport was optimum at pH 7.6-8.0. In addition, prior exposure of the cells to elevated extracellular Pi (2-3 mM) strongly enhanced subsequent Pi uptake, which appeared to depend on prior loading of the cells with mineral ions. Prevention of Pi loading by pretreatment with Pi transport inhibitors not only inhibited subsequent cellular Pi uptake, it also blocked mineral formation. Treatment with elevated extracellular Pi did not induce apoptosis in these GP chondrocytes.