Yinlong Cai

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)-[3H]HETE (Kd = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC50 = 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.

Role for β3 integrins in human melanoma growth and survival

The role of αIIbβ3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of αIIbβ3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that αIIbβ3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of αIIbβ3 was 8% (±1.8), 33% (±10.4) and 62% (±5) in melanomas ranging in thickness from 0–1.5 mm, 1.5–4.0 mm and >4 mm, respectively; αvβ3 was equally high all categories. To determine biological function, we stably transfected αIIbβ3 into human melanoma cells that express αvβ3, but not αIIbβ3. Surface expression of αvβ3 remained unaltered between αIIbβ3 (+) and mock transfected counterparts. The αIIbβ3 (+) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of αIIbβ3 displaced αvβ3 from focal contact points. When implanted subcutaneously into SCID mice, the αIIbβ3 (+) cells developed ∼4‐fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co‐expression of the 2 β3 integrins, αvβ3 and αIIbβ3, in human melanoma cells enhanced cell survival and promoted growth in vivo.

Role of αIIbβ3 integrin in prostate cancer metastasis

Integrins participate in cell‐cell and cell‐matrix interactions. In this study we determined whether αIIbβ3 integrin is involved in metastasis of human prostate adenocarcinoma cells.

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.

12-Lipoxygenase and the regulation of hypoxia-inducible factor in prostate cancer cells

12-Lipoxygenase, an arachidonic acid metabolizing enzyme of the lipoxygenase pathway, has been implicated as a major factor in promoting prostate cancer progression and metastasis. The ability of 12-LOX to aggravate the disease was linked to its proangiogenic role. Recent studies clearly demonstrated that 12-LOX enhances the expression and secretion of the angiogenic factor, vascular endothelial growth factor (VEGF) thus providing a direct link between this enzyme and its angiogenic properties. In the present study we have investigated the relationship between 12-LOX and hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor involved in the regulation of VEGF expression under hypoxic conditions in solid tumors. Our findings have revealed that HIF-1 is one of the target transcription factors regulated by 12-LOX and 12(S)-HETE, in hypoxic tumor cells of the prostate. Regulation of HIF-1alpha by 12-LOX adds to the complexity of pathways mediated by this enzyme in promoting prostate cancer angiogenesis and metastasis. We have evidence that 12-LOX increases the protein level, mRNA, and functional activity of HIF-1alpha under hypoxic conditions, one of the mechanisms by which it upregulates VEGF secretion and activity.

Convergence of eicosanoid and integrin biology: 12-lipoxygenase seeks a partner.

BackgroundIntegrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase (12-LOX, ALOX12), we identified the integrin β4 (ITGB4).MethodsUsing a cultured mammalian cell model, we have demonstrated that ITGB4 stimulation leads to recruitment of 12-LOX from the cytosol to the membrane where it physically interacts with the integrin to become enzymatically active to produce 12(S)-HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes.ResultsThe net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of β4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX.ConclusionsThis is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated β4 integrin. Integrin β4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin β4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association.

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. FASEB J. 30, 2360–2369 (2016). www.fasebj.org

A naturally occurring truncated β3 integrin in tumor cells: Native anti-integrin involved in tumor cell motility

Alternatively spliced integrins may play an important role in integrin mediated tumor cell adhesion, spreading, and migration. Here we report in human tumor cells a naturally occurring alternatively spliced variant of the β3 integrin [i.e. truncated (tr) β3] that lacked a cytoplasmic and a transmembrane domain. The presence of tr-β3 was demonstrated at the mRNA level by RT-PCR, cloning, and sequencing; at the protein level by immunohistochemistry and Western Blotting. The alternately spliced β3 integrin was detected in human prostate carcinomas, breast carcinomas, and melanoma cells. Expression in vivo was confirmed by immunohistochemistry with an antibody to tr-β3 that does not recognize wild type β3. Tumor cells secreted this protein and deposited it on the extracellular matrix. Secreted tr-β3 inhibited adhesion of melanoma and prostate cancer cells to fibronectin and vitronectin, which was partially reversed by adsorption of tr-β3 from the media. Confocal microscopy and time lapse live cell microscopy demonstrated that tr-β3 distributed to the trailing edge of migrating cells, which may represent an alternative cell detachment mechanism in these cells. Results suggest that tr-β3 may act as an anti-integrin and play a crucial role in cell migration, which is an important process in tumor invasion and metastasis.

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

ABSTRACT 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 &bgr;4 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 &bgr;4 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 &bgr;4‐integrin‐mediated phosphorylation of 12‐LOX. HIGHLIGHTSITGB4 ligation in A431 cells regulates Src phosphorylation/activation.12LOX enzymatic activity is increased by ITGB4‐induced pSrc phosphorylation.Tyrosine residues 19 and 614 in 12LOX are pSrc targets post ITGB4 ligation.Phosphorylation state of 12LOX Y19 and Y614 influences cancer phenotypes.

Abstract 2890: A novel interaction between 12-lipoxygenase and integrin subunit Beta4 plays a role in tumor migration, invasion and metastasis

This study has the potential to establish a new paradigm in adhesion-mediated control of eicosanoid production and could be relevant to tumor migration, invasion, and metastasis. 12-Lipoxygenase metabolizes arachidonic acid to form 12(S)-HETE, a metabolite known to play a significant role in the process of tumor-induced angiogenesis and metastasis. The role of 12-LOX in proliferation, survival, and motility of tumor cells as well as in angiogenesis has been described for a variety of cancers. Similarly Alpha6Beta4 integrin also plays a role in both angiogenesis and tumorigenesis in addition to having a role in the formation of hemidesmosomes (HD). Evidence suggests a correlation between 12-LOX and integrin Beta4 since they play a similar role in cancer progression. The physical interaction between 12-LOX and Beta4 was first identified by a yeast two-hybrid screen using 12-LOX as bait against a library made from A431 cells that are known to have endogenous expression of both proteins. We verified this interaction by co-immunoprecipitation from A431 cells, and recapitulated it in CHO and PC-3 cells ectopically expressing 12-LOX and the cytoplasmic tail of Beta4 integrin. Additionally our data shows that ligation or clustering of Alpha6Beta4 integrin by the natural ligand laminin or an antibody mimic of laminin, 3E1, caused an increase in translocation of 12-LOX from the cytosol to membrane and an increase in 12-LOX enzymatic activities as assayed by immunofluorescence and 12(S)-HETE measurements respectively. 3E1-stimulated A431 cells showed increased cell migration but there was 40% decrease in presence of 12-LOX inhibitor BMD122 which confirms the role of 12-LOX in cell invasion. Here we describe for the first time the interaction domains that unite Beta4 integrin and 12-LOX. We mapped the domains in the cytoplasmic tail of Beta4 critical for interacting with 12-LOX and found that the 1126-1157 domain in the cytoplasmic tail of Beta4 is important for binding of 12-LOX. Also we have shown the mutant fragment (1126-1315) interacts with 12-lipoxygenase in the cytosol and prevent its binding to native, full-length membrane-associated Beta4 and thereby prevents 12-LOX activation. This physical interaction has functional implications with regard to decreased cell survival and migration. Determination of the structural basis for protein-protein interaction may provide potential means to manipulate or block such interaction, which may yield therapeutic benefits. We propose that the results obtained from these studies will provide a novel, and important dimension to both integrin and eicosanoid biology. Acknowledgements: This project is supported by NIH: NCI, 2 R01 CA029997-17A1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2011-2890