5-Oxo-ETE/OXER1: A Link between Tumor Cells and Macrophages Leading to Regulation of Migration.

Chronic inflammation is an important factor in the development of cancer. Macrophages found in tumors, known as tumor associated macrophages (TAMs), are key players in this process, promoting tumor growth through humoral and cellular mechanisms. 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), an arachidonic acid metabolite, has been described to possess a potent chemoattractant activity for human white blood cells (WBCs). The biological actions of 5-oxo-ETE are mediated through the GPCR 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid receptor (OXER1). In addition, we have previously reported OXER1 as one of the membrane androgen receptors with testosterone antagonizing 5-oxo-ETE's actions. OXER1 is highly expressed in inflammatory cells and many normal and cancer tissues and cells, including prostate and breast cancer, promoting cancer cell survival. In the present study we investigate the expression and role of OXER1 in WBCs, THP-1 monocytes, and THP-1 derived macrophages, as well as its possible role in the interaction between macrophages and cancer cells (DU-145 and T47D). We report that OXER1 is differentially expressed between WBCs and macrophages and that receptor expression is modified by LPS treatment. Our results show that testosterone and 5-oxo-ETE can act in an antagonistic way affecting Ca2+ movements, migration, and cytokines' expression in immune-related cells, in a differentiation-dependent manner. Finally, we report that 5-oxo-ETE, through OXER1, can attract macrophages to the tumor site while tumor cells' OXER1 activation in DU-145 prostate and T47D breast cancer cells, by macrophages, induces actin cytoskeletal changes and increases their migration.

Nuclear translocation of the membrane oxoeicosanoid/androgen receptor, OXER1: Possible mechanisms involved.

OXER1, the receptor for the arachidonic acid metabolite 5-οxo-eicosatetraenoic acid (5-oxo-ETE), has been reported to also bind and mediate the membrane-initiated actions of androgens. Indeed, androgens antagonize the 5-oxo-ETE effects through OXER1, affecting a number of signaling pathways and inhibiting cancer cell proliferation and migration. OXER1, being a GPCR, was classically described to be localized in the plasma membrane. However, for numerous GPCRs, there is now strong evidence that they can be also found in other cellular compartments, including the nucleus. The aim of the present work was to investigate OXER1's possible localization in the nucleus and identify the mechanism(s) involved. For this purpose, we verified OXER1's nuclear presence by immunofluorescence and western blot, in whole cells and nuclei of two different prostate cancer cell lines (DU-145 and LNCaP) and in CHO cells transfected with a GFP labelled OXER1, both in untreated and OXER1 ligands' treated cells. Mutated, OXER1-tGFP expressing, CHO cells were used to verify that OXER1 agonist (5-oxo-ETE) binding is necessary for OXER1 nuclear translocation. NLS sequences were in silico identified, and a specific inhibitor, as well as, specific importins' siRNAs were also utilized to explore the mechanism involved. Moreover, we examined the role of palmitoylation in OXER1 nuclear translocation by in silico identifying possible palmitoylation sites and using a palmitoylation inhibitor. Our results clearly show that OXER1 can be localized in the nucleus, in an agonist-dependent manner, that is inhibited by androgens. We also provide evidence for two possible mechanisms for its nuclear trafficking, that involve receptor palmitoylation and importin-mediated cytoplasmic-nuclear transport. In our knowledge, it is the first time that a membrane androgen receptor is identified into the nucleus, suggesting an alternative, more direct, mode of action, involving nuclear mechanisms. Therefore, our findings provide new insights on androgen-mediated actions and androgen-lipid interactions, and reveal new possible therapeutic targets, not only for cancer, but also for other pathological conditions in which OXER1 may have an important role.

Promyelocytic leukemia protein regulates angiogenesis and epithelial-mesenchymal transition to limit metastasis in MDA-MB-231 breast cancer cells.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with the suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed the induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth, and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Promyelocytic leukemia protein (PML) and stem cells: from cancer to pluripotency.

The promyelocytic leukemia protein (PML) is the core organizer of cognate nuclear bodies (PML-NBs). Through physical interaction or modification of diverse protein clients, PML-NBs regulate a multitude of - often antithetical- biological processes such as antiviral and stress response, inhibition of cell proliferation and autophagy, and promotion of apoptosis or senescence. Although PML was originally recognized as a tumor-suppressive factor, more recent studies have revealed a "double-faced" agent role for PML. Indeed, PML displayed tumor cell pro-survival and pro-migratory functions via inhibition of migration suppressing molecules or promotion of transforming growth factor beta (TGF-ß) mediated Epithelial-Mesenchymal Transition (EMT) that may promote cancer cell dissemination. In this line, PML was found to correlate with poor patient prognosis in distinct tumor contexts. Furthermore, in the last decade, a number of publications have implicated PML in the physiology of normal or cancer stem cells (CSCs). Promyelocytic leukemia protein activates fatty acid oxidation (FAO), a metabolic mechanism required for the asymmetric divisions and maintenance of hematopoietic stem cells (HSCs). In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PML is required for maintenance of the naïve and acquisition of the induced pluripotency state, respectively. Correspondingly, PML ablation causes significant morphological gene expression and lineage choice changes. In this review, we focus on the mechanisms orchestrated by PML and PML-NBs in cancer and healthy stem cells, from cell physiology to the regulation of chromatin dynamics.

Phytochemical and genetic characterization of styles of wild Crocus species from the island of Crete, Greece and comparison to those of cultivated C. sativus.

The aim of this study was to contribute to the characterization of Crocus taxa using morphological, phytochemical and genetic analysis. The styles of C. cartwrightianus, C. oreocreticus and C. laevigatus, collected in the island of Crete were compared to those of C. sativus cultivated at the region of Western Macedonia (Greece). Phytochemical analysis was done using GC-MS and HPLC methods, while ISSR markers were used for their genetic characterization. Safranal was the major volatile component of the styles of C. sativus, 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde of C. cartwrightianus and C. oreocreticus, and isophorone of C. laevigatus. C. sativus had the highest content of crocins and picrocrocin, while C. laevigatus the lowest (only 5% of C. sativus' quantity) and negligible amount of picrocrocin. According to both the genetic and the chemical analysis, C. cartwrightianus is more related to C. oreocreticus, while C. sativus and C. laevigatus are more distinct. Concordance between the two different types of data was also confirmed by the Mantel test (r = 0.932, P = .68). This is the first thorough screening of secondary metabolites (volatile and non-volatile) and also genetic and morphological characters of wild Crocus styles simultaneously, that contributes to the identification and valorisation of genotypes with similar to C. sativus traits which may be introduced as new cultivars through breeding.