Human Cancer Biology Comparative Analysis of Peritoneum and Tumor Eicosanoids and Pathways in Advanced Ovarian Cancer Ralph S. Freedman,1Ena Wang,5 SoniaVoiculescu,5 Rebecca Patenia,1Roland L. Bassett, Jr.,2 Michael Deavers,3 Francesco M. Marincola,5 PeiyingYang,4 and Robert A. Newman4 Abstract Purpose: To describe the eicosanoid profile and differentially expressed eicosanoid and arachidonic acid pathway genes in tissues from patients with advanced epithelial ovarian cancer (EOC). Experimental Design:We first employed electrospray tandem mass spectrometry to determine tissue-specific concentrations of the eicosanoids prostaglandin E2 (PGE2), the hydroxyeicosate- traenoic acids (12-HETE and 5-HETE), and leukotriene (LTB4), selected for tumor growth potential, and two other bioactive lipids (15-HETE and 13-HODE) with tumor cell proliferation interference potential. The cellular location of eicosanoid activity was identified by immunofluo- rescence antibody costaining and confocal microscopy. Differential analysis of eicosanoid and arachidonic pathway genes was done using a previously validated cDNA microarray platform. Tissues usedincluded EOC tumor, tumor-free malignant peritoneum (MP),and benignperitoneum (BP) from patients with benign pelvic disease. Results: (a) Eicosanoid products were detected in tumor, MP, and BP specimens. PGE2 levels were significantly elevated in tumors in an overall comparison with MP or BP (P < 0.001). Combined levels of PGE2, 12-HETE, 5-HETE, and LTB4 increased progressively from low to high concentrations in BP, MP, and tumors (P = 0.012). Neither 15-HETE nor 13-HODE showed a significant opposite trend toward levels found in BP. (b) Tissue specimens representing common EOC histotypes showed strong coexpressions of cyclooxygenases (COX-1) and prostaglandin E synthases (PGES-1) on tumor cells, whereas intratumoral or peritumoral MO/MA coexpressed COX-1and COX-2and PGES-1and PGES-2,respectively. ( c)cDNAmicroarrayanalysisofMP, BP, and tumor showed that a number of eicosanoid and arachidonic acid pathway genes were differentially expressed in MP and BP compared with tumor, except for CYP2J2, which was increased in tumors. Conclusions: Elevated levels of eicosanoid metabolites in tumors and differential expression of eicosanoid and arachidonic acid pathway genes in the peritoneum support the involvement of bioactive lipids in the inflammatory tumor environment of EOC. Eicosanoids is a collective term for oxygenated derivatives of Arachidonic acid liberated by PLA2 is metabolized by cyclo- different 20-carbon fatty acids such as leukotrienes and oxygenase (COX) and lipoxygenase (LOX) enzymes variously prostanoids (prostaglandins, prostacyclins, and thrombox- expressed in cells and tissues, and contribute to the production anes). These bioactive lipids have important functional roles of specific metabolites (1, 3, 4). These bioactive lipids or in regulating many physiologic processes and inflammatory eicosanoids then exert their biological effects in an autocrine or responses (1). Eicosanoid production is a tightly regulated paracrine manner by binding to specific G-coupled receptors process that depends on (a) the acylation and transfer of (5–8). In a previous study, we showed that sPLA2 (group 2a) arachidonic acid into specific phospholipid pools by arach- gene transcripts in tumor-free specimens from the malignant idonic acid–selective acyltransferase and transacylase reactions peritoneum (MP) of patients with epithelial ovarian cancer and (b) the release of these pools by a variety of phospholipase (EOC) were differentially expressed when compared with those A (PLA ) enzymes (2). in tumor tissue (9). We also verified the expression of PLA2 at 2 2 the protein level in MO/MA in ascitic specimens (10). MO/MA are the most prominent population of inflammatory cells 1 2 observed in the tumor and peritoneal microenvironment of Authors’Affiliations: Departments of Gynecologic Oncology, Quantitative EOC (10, 11). Sciences, 3Pathology, and 4Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, and 5Immunogenetics Section, Despite the well-described roles of eicosanoids in cancer Department ofTransfusion Medicine, NIH, Bethesda, Maryland inhibition or proliferation, few studies have focused on tissue- Received 3/9/07; revised 5/16/07; accepted 7/13/07. specific eicosanoid metabolism, largely because methods for Requests for reprints: Ralph S.Freedman,The University of Texas M. D. Anderson identifying and quantifying multiple COX- and LOX-derived Cancer Center, P.O. Box 301439, Unit 1362, Houston,TX 77230. Phone: 713-792- 2764; Fax: 713-792-7586; E-mail: [email protected]. products in specific tissues were lacking. One of the coauthors F 2007 American Association for Cancer Research. (R.A. Newman) has established an analytic procedure for this doi:10.1158/1078-0432.CCR-07-0583 purpose based on liquid chromatography/electrospray tandem Clin Cancer Res 2007;13(19) October 1, 2007 5736 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Eicosanoid and Arachidonic Acid Pathways in Ovarian Cancer mass spectrometry analysis (12, 13). This lipidomics technique 15-LOX1). Eicosanoid levels were compared between groups using is currently being used to identify and measure specific changes ANOVA and t tests. Some analyses were repeated using Kruskal-Wallis associated with endogenous COX and LOX activities in cells and Wilcoxon tests; results were similar and are not reported in this P and tissues; such changes are thought to reflect alterations in article. Statistical significance was declared at < 0.05. No adjustment was made for the multiplicity of testing. eicosanoid profiles between normal, inflamed, and malignant Fluorescence labeled multi-antibody costaining and confocal microscopy tissues. These studies have shown significant differences in visualization of peritoneal and tumor biopsy specimen cells. Our method patterns of eicosanoid metabolism between different types to prepare tissues for staining and reading of stained slides are fully of malignant tissues; for example, it has been shown that described elsewhere (10). A sequential staining technique was used as colon cancer is regulated in part by the relative expression of follows: 3 h incubation with the first primary antibody (red) at room 13-S-hydroxyoctadecadienoic acid (13-S-HODE; ref. 14), temperature, overnight incubation with the second primary anti- that glioblastomas invariably overexpress 5-LOX,6 and that body (blue/green) at 4jC, Secondary antibodies were incubated with prostate cancer is connected to an age-associated decline in cryostat-prepared sections for 1h after the incubations with the primary the expression of 15-hydroxyeicosatetraenoic acid (15-HETE), antibodies were complete. Nonspecific binding was blocked by adding a tumor suppressor (15), as well as up-regulation of 12-LOX 5% normal goat serum for 30 min. The primary antibodies used were COX-1 monoclonal mouse antibody (12E12), IgM, 1:25 dilution which, in turn, inhibits Rb tumor suppressor activity (16). (GeneTex, Inc.); COX-2 mouse monoclonal antibody, IgG1, 1:50 In the current study, we employed electrospray tandem mass dilution (Cayman Chemical Co.); prostaglandin E synthase-1(PGES-1; spectrometry analysis combined with gene expression analysis microsomal) polyclonal rabbit antibody, IgG, 1:50 dilution (Cayman); to produce the first description of specific variations in prostaglandin E synthase-2 (PGES-2; microsomal) polyclonal rabbit eicosanoid products and enzymes involved with eicosanoid antibody, IgG, 1:50 dilution (Cayman); 15-LOX2, 15-LOX2 polyclonal and arachidonic acid pathways in ovarian tumor tissues. Our rabbit antibody, IgG, 1:50 dilution (Cayman); 5-LOX polyclonal findings show that endogenous levels of prostaglandin E2 rabbit antibody, IgG, 1:50 dilution (Cayman); mouse anti-human CD163, IgG , 1:200 dilution (Serotec); mouse anti-human cytokeratin (PGE2), 5-HETE, and 12-HETE increase progressively from 1 n benign peritoneum (BP) tissue, through EOC peritoneum clone AE1/AE3, IgG1, , 1:100 dilution (DAKOCytomation). Second- (MP), to EOC tumor. Distribution of the expressive enzymes ary antibodies were Cy3 (red)-conjugated affinipure goat anti-mouse IgM, A chain specific; Cy3 (red)-conjugated affinipure goat anti-mouse involved in these pathways at the cellular and gene transcript IgG, Fcg subclass 1specific; Cy3 (red) conjugated affinipure goat anti- levels is shown. Differential gene expression profiles involving rabbit IgG (H+L); Cy2 (green)-conjugated affinipure goat anti-mouse eicosanoid and arachidonic acid metabolism pathways were IgG1,Fcg subclass 1–specific; Cy5 (blue)-conjugated affinipure goat shown by cDNA microarray analysis. anti-mouse IgG, Fcg subclass 1specific (all from Jackson Immuno- Research Labs). When two unconjugated primary antibodies from the same host Materials and Methods species and the same class of immunoglobulin IgG1 were used, any open antigen binding sites on the first and secondary antibodies were Electrospray tandem mass spectrometry analyses. Endogenous levels saturated with 5% normal mouse serum. Mouse immunoglobulins are of key eicosanoids from cells and tissues were measured by validated, sterically covered with monovalent affinipure Fab fragment goat anti- published methods (12, 17). Fresh-frozen tumor and generally matched mouse IgG (H+L), 1:65 dilution (Jackson ImmunoResearch Labs, Inc.).