comparativeanalysis of peritoneum andtumor eicosanoids and ... · comparativeanalysis of peritoneum...

Comparative Analysis of Peritoneum andTumor Eicosanoidsand Pathways in Advanced Ovarian CancerRalph S. Freedman,1EnaWang,5 SoniaVoiculescu,5 Rebecca Patenia,1Roland L. Bassett, Jr.,2

Michael Deavers,3 FrancescoM.Marincola,5 PeiyingYang,4 and Robert A. Newman4

Abstract Purpose: To describe the eicosanoid profile and differentially expressed eicosanoid andarachidonic acid pathway genes in tissues from patients with advanced epithelial ovarian cancer(EOC).Experimental Design:We first employed electrospray tandemmass spectrometry to determinetissue-specific concentrations of the eicosanoids prostaglandin E2 (PGE2), the hydroxyeicosate-traenoic acids (12-HETE and 5-HETE), and leukotriene (LTB4), selected for tumor growthpotential, and two other bioactive lipids (15-HETE and 13-HODE) with tumor cell proliferationinterference potential. The cellular location of eicosanoid activity was identified by immunofluo-rescence antibody costaining and confocal microscopy. Differential analysis of eicosanoid andarachidonic pathway genes was done using a previously validated cDNA microarray platform.Tissuesused includedEOC tumor, tumor-freemalignant peritoneum (MP), andbenignperitoneum(BP) frompatientswithbenignpelvic disease.Results: (a) Eicosanoid products were detected in tumor, MP, and BP specimens. PGE2 levelswere 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 highconcentrations in BP, MP, and tumors (P = 0.012). Neither 15-HETE nor 13-HODE showed asignificant opposite trend toward levels found in BP. (b) Tissue specimens representing commonEOC histotypes showed strong coexpressions of cyclooxygenases (COX-1) and prostaglandin Esynthases (PGES-1) on tumor cells, whereas intratumoral or peritumoral MO/MA coexpressedCOX-1and COX-2 and PGES-1and PGES-2, respectively. (c) cDNA microarray analysis of MP,BP, and tumor showed that a number of eicosanoid and arachidonic acid pathway genes weredifferentially expressed in MP and BP compared with tumor, except for CYP2J2, which wasincreased in tumors.Conclusions: Elevated levels of eicosanoid metabolites in tumors and differential expression ofeicosanoid and arachidonic acid pathway genes in the peritoneum support the involvement ofbioactive lipids in the inflammatory tumor environment of EOC.

Eicosanoids is a collective term for oxygenated derivatives ofdifferent 20-carbon fatty acids such as leukotrienes andprostanoids (prostaglandins, prostacyclins, and thrombox-anes). These bioactive lipids have important functional rolesin regulating many physiologic processes and inflammatoryresponses (1). Eicosanoid production is a tightly regulatedprocess that depends on (a) the acylation and transfer ofarachidonic acid into specific phospholipid pools by arach-idonic acid–selective acyltransferase and transacylase reactionsand (b) the release of these pools by a variety of phospholipaseA2 (PLA2) enzymes (2).

Arachidonic acid liberated by PLA2 is metabolized by cyclo-oxygenase (COX) and lipoxygenase (LOX) enzymes variouslyexpressed in cells and tissues, and contribute to the productionof specific metabolites (1, 3, 4). These bioactive lipids oreicosanoids then exert their biological effects in an autocrine orparacrine manner by binding to specific G-coupled receptors(5–8). In a previous study, we showed that sPLA2 (group 2a)gene transcripts in tumor-free specimens from the malignantperitoneum (MP) of patients with epithelial ovarian cancer(EOC) were differentially expressed when compared with thosein tumor tissue (9). We also verified the expression of PLA2 atthe protein level in MO/MA in ascitic specimens (10). MO/MAare the most prominent population of inflammatory cellsobserved in the tumor and peritoneal microenvironment ofEOC (10, 11).Despite the well-described roles of eicosanoids in cancer

inhibition or proliferation, few studies have focused on tissue-specific eicosanoid metabolism, largely because methods foridentifying and quantifying multiple COX- and LOX-derivedproducts in specific tissues were lacking. One of the coauthors(R.A. Newman) has established an analytic procedure for thispurpose based on liquid chromatography/electrospray tandem

Human Cancer Biology

Authors’ Affiliations: Departments of 1Gynecologic Oncology, 2QuantitativeSciences, 3Pathology, and 4Experimental Therapeutics, The University of TexasM. D. Anderson Cancer Center, Houston,Texas, and 5Immunogenetics Section,Department ofTransfusionMedicine, NIH, Bethesda, MarylandReceived 3/9/07; revised 5/16/07; accepted 7/13/07.Requests for reprints:RalphS.Freedman,TheUniversity ofTexasM.D.AndersonCancer Center, P.O. Box 301439, Unit 1362, Houston,TX 77230. Phone: 713-792-2764; Fax: 713-792-7586; E-mail: [email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-07-0583

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mass spectrometry analysis (12, 13). This lipidomics techniqueis currently being used to identify and measure specific changesassociated with endogenous COX and LOX activities in cellsand tissues; such changes are thought to reflect alterations ineicosanoid profiles between normal, inflamed, and malignanttissues. These studies have shown significant differences inpatterns of eicosanoid metabolism between different typesof malignant tissues; for example, it has been shown thatcolon cancer is regulated in part by the relative expression of13-S -hydroxyoctadecadienoic acid (13-S-HODE; ref. 14),that glioblastomas invariably overexpress 5-LOX,6 and thatprostate cancer is connected to an age-associated decline inthe expression of 15-hydroxyeicosatetraenoic acid (15-HETE),a tumor suppressor (15), as well as up-regulation of 12-LOXwhich, in turn, inhibits Rb tumor suppressor activity (16).In the current study, we employed electrospray tandem mass

spectrometry analysis combined with gene expression analysisto produce the first description of specific variations ineicosanoid products and enzymes involved with eicosanoidand arachidonic acid pathways in ovarian tumor tissues. Ourfindings show that endogenous levels of prostaglandin E2(PGE2), 5-HETE, and 12-HETE increase progressively frombenign peritoneum (BP) tissue, through EOC peritoneum(MP), to EOC tumor. Distribution of the expressive enzymesinvolved in these pathways at the cellular and gene transcriptlevels is shown. Differential gene expression profiles involvingeicosanoid and arachidonic acid metabolism pathways wereshown by cDNA microarray analysis.

Materials and Methods

Electrospray tandem mass spectrometry analyses. Endogenous levelsof key eicosanoids from cells and tissues were measured by validated,published methods (12, 17). Fresh-frozen tumor and generally matchedperitoneum samples were obtained from patients with EOC and pelvicperitoneum samples from patients with benign pelvic disease and EOCtumors. All specimens were obtained under an Institutional ReviewBoard–approved protocol. MP specimens with contaminating tumor asidentified by a gynecologic oncology pathologist (M. Deavers) wereexcluded from eicosanoid and microarray analyses. Specimens foreicosanoid analyses were obtained in the operating room, snap-frozendirectly, and stored at -80jC until analyzed. Endogenous eicosanoidswere measured with a Quattro Ultima tandem mass spectrometer(Micromass) equipped with an Agilent HP1100 binary high-pressureliquid chromatography inlet. Eicosanoids were separated on a Luna 5APhenyl-Hexyl 2 � 150 mm column (Phenomenex) with a rapid lineargradient of 70% to 90% methanol in 4 min over 10 min. The mobilephase consisted of 10 mmol/L of ammonium acetate (pH 8.5) andmethanol (30:70). Fragmentation of all compounds was achieved byusing argon as the collision gas. This method produces excellentlinearity and a lower limit of quantification for eicosanoids of 1 ng/mL,which is adequate to assess endogenous eicosanoid metabolism thatcommonly occurred as ng/mg of protein in 5 � 106 cell aliquots ortissue samples. We have used this method to characterize in detaileicosanoid metabolism in human lung, prostate, breast, and colontissues as well as in numerous cell lines. The following eicosanoids weremeasured: PGE2 (product of cyclooxygenase pathway), 5-HETE(product of 5-lipoxygenase or 5-LOX), LTB4 (product of leukotrienes-A4 hydrolase and downstream product of 5-LOX), 12-HETE (product of12-LOX), 15-HETE (product of 15-LOX2), and 13-HODE (product of

15-LOX1). Eicosanoid levels were compared between groups usingANOVA and t tests. Some analyses were repeated using Kruskal-Wallisand Wilcoxon tests; results were similar and are not reported in thisarticle. Statistical significance was declared at P < 0.05. No adjustmentwas made for the multiplicity of testing.Fluorescence labeled multi-antibody costaining and confocal microscopy

visualization of peritoneal and tumor biopsy specimen cells. Our methodto prepare tissues for staining and reading of stained slides are fullydescribed elsewhere (10). A sequential staining technique was used asfollows: 3 h incubation with the first primary antibody (red) at roomtemperature, overnight incubation with the second primary anti-body (blue/green) at 4jC, Secondary antibodies were incubated withcryostat-prepared sections for 1 h after the incubations with the primaryantibodies were complete. Nonspecific binding was blocked by adding5% normal goat serum for 30 min. The primary antibodies used wereCOX-1 monoclonal mouse antibody (12E12), IgM, 1:25 dilution(GeneTex, Inc.); COX-2 mouse monoclonal antibody, IgG1, 1:50dilution (Cayman Chemical Co.); prostaglandin E synthase-1 (PGES-1;microsomal) polyclonal rabbit antibody, IgG, 1:50 dilution (Cayman);prostaglandin E synthase-2 (PGES-2; microsomal) polyclonal rabbitantibody, IgG, 1:50 dilution (Cayman); 15-LOX2, 15-LOX2 polyclonalrabbit antibody, IgG, 1:50 dilution (Cayman); 5-LOX polyclonalrabbit antibody, IgG, 1:50 dilution (Cayman); mouse anti-humanCD163, IgG1, 1:200 dilution (Serotec); mouse anti-human cytokeratinclone AE1/AE3, IgG1, n, 1:100 dilution (DAKOCytomation). Second-ary antibodies were Cy3 (red)-conjugated affinipure goat anti-mouseIgM, A chain specific; Cy3 (red)-conjugated affinipure goat anti-mouseIgG, Fcg subclass 1 specific; Cy3 (red) conjugated affinipure goat anti-rabbit IgG (H+L); Cy2 (green)-conjugated affinipure goat anti-mouseIgG1, Fcg subclass 1–specific; Cy5 (blue)-conjugated affinipure goatanti-mouse IgG, Fcg subclass 1 specific (all from Jackson Immuno-Research Labs).

When two unconjugated primary antibodies from the same hostspecies and the same class of immunoglobulin IgG1 were used, anyopen antigen binding sites on the first and secondary antibodies weresaturated with 5% normal mouse serum. Mouse immunoglobulins aresterically covered with monovalent affinipure Fab fragment goat anti-mouse IgG (H+L), 1:65 dilution (Jackson ImmunoResearch Labs, Inc.).Negative controls employed secondary antibodies alone.

Tissue sections were mounted with Slow-Fade Gold Anti-Fadereagent (Molecular Probes) and viewed with an Olympus FV500 laserscanning confocal microscope; images were captured at 400� and600� magnification by Fluoview software Version 4.3.Differential gene expression analysis. Specimen collection and

sample preparation were as described previously (9) but with asubstantially increased sample size. Specimens for microarray analyseswere transferred directly to the laboratory from the operating room incold saline, then snap-frozen in RNAlate (Ambion, Inc.), stored at-80jC. Processing of frozen material and extraction of RNA with qualitycontrols were previously described (9). Among the specimens, 15 ofthem were from peritoneum associated with benign pelvic disease,designated ‘‘BP’’, 35 specimens from EOC or histopathologically relatedtumors, designated ‘‘Tu’’, and 27 tumor-free peritoneal specimensdesignated ‘‘MP.’’ Specimens that had microscopic tumor invasion insubsequent histopathologic examination were excluded. Total RNAisolation, RNA amplification and array hybridization were done asdescribed (18). Custom microarrays were printed at the Immunoge-netics Section, Department of Transfusion Medicine, Clinical Center,NIH with a configuration of 32 � 24 � 23 and containing 17,500elements. For a complete list of genes included in the Hs-CCDTM-17.5k-1px, printing is available at our web site.7 Genes involved ineicosanoid and arachidonic acid pathways from the cDNA microarraydata set were selected according to the KEGG pathway finder8 from the

6 R.A. Newman, P. Yang, unpublished data.

7 http://nciarray.nci.nih.gov/gal_files/index.shtml8 http://www.genome.jp/kegg/pathway.html

Eicosanoid and Arachidonic Acid Pathways in Ovarian Cancer

www.aacrjournals.org Clin Cancer Res 2007;13(19) October1, 20075737



complete cDNA microarray data set after normalization and back-ground subtraction, using BRB array tool.9 Two-sample t tests weredone between MP versus BP, MP versus tumor and BP versus tumor. Inaddition, F tests (n = 3) were also done with 10,000 randompermutations using the BRB array tool. Genes with P < 0.05 weredefined as significantly differentially expressed genes and werevisualized by cluster and TreeView analysis. A selected gene group wasused in the analysis.

Results

Eicosanoid metabolites increased in EOC tumor, MP, and BPtissues. Eicosanoid metabolite levels were measured on frozentumor specimens obtained from 12 chemotherapy-naBve

patients with EOC. Eleven of 12 patients in the malignantgroup had stage III or IV carcinomas with serous components.Patients with benign pathology included entities such as benignovarian epithelial or germ cell tumors (two patients), lowmalignant potential tumors of the ovary without invasiveimplants (four patients), chronic pelvic inflammatory disease(two patients). Median ages were 68 (range, 61-80) for themalignant group and 62 (range, 28-78) for the benign group.There were eight matched specimens of tumor and MPperitoneum and separate MP or tumor specimens from fourpatients, and BP was obtained from eight patients with a varietyof benign pelvic conditions who were scheduled for pelvicabdominal surgery. Only MP specimens that were tumor-freewere used.Quantitative eicosanoid analysis of tumor, MP or BP tissues

were analyzed for PGE2, 5-HETE, 12-HETE, LTB4, 15-HETE,and 13-HODE, and results are shown in Fig. 1 and Table 1,expressed in nanograms of eicosanoid per milligram of tissueprotein. PGE2, an eicosanoid commonly associated withmalignant disease, was significantly elevated in tumor com-pared with either MP (P = 0.003) or BP (P = 0.004; Fig. 1A).When PGE2, 5-HETE, 12-HETE, and LTB4 (which are metabo-lites more clearly identified with tumor cell proliferation) areaveraged, they show a significant linear trend upward from BPthrough EOC peritoneum and EOC tumor (P = 0.012; Fig. 1B).Other individual comparisons were not statistically significant(Table 1). Although mean values for the 15-LOX2 product, 15-HETE, and the 15-LOX1 product, 13-HODE, seemed to movein the opposite direction, the trends were not statisticallysignificant (Fig. 1; Table 1).Immunocostaining of eicosanoid pathway enzymes. We next

examined coexpression of eicosanoid products by IIF usingconfocal microscopy on: tumor tissue, peritoneum of EOCpatients, and peritoneum from patients with benign pelvicdisease on two to three individual patients. We showed, asothers have (19), that COX-1 expression was strongly expressedon the tumor cells, whereas COX-2 was only weakly expressedon tumor cell islets. In addition, expression of PGES-1 andPGES-2 paralleled the results for COX-1 and COX-2 with weakexpression of PGES-2 (Fig. 2). In contrast, COX-1, COX-2, and

9 http://linus.nci.nih.gov/BRB-ArrayTools.html

Fig. 1. Boxplots of median, quartiles, and range for level of eicosanoids PGE2,LTB4, 15-HETE,12-HETE, 5-HETE, and13-HODE in BP, MP, and tumor tissues.A, individual eicosanoid values; B, average combined PGE2, LTB4, 12-HETE, and5-HETE values.

Table 1. Overall and individual tissue comparisonof eicosanoid values in BP, MP, and tumor tissues

Variables Comparison of meaneicosanoid values (ng/mg)

BP (N) MP (N) Tumor

PGE2 1.14 (8)* 1.60 (10)c 6.86 (10)5-HETE 0.56 (8) 1.10 (10) 2.07 (10)12-HETE 1.95 (8) 4.37 (10) 5.60 (10)LTB4 0.57 (8) 1.68 (10) 0.86 (10)15-HETE 16.05 (8) 6.55 (10) 6.85 (10)13-HODE 4.62 (8) 4.88 (10) 2.52 (10)

NOTE: Mean values were from eight pairs of BP samples and fromone to two samples/patients obtained from 10 MP and from 10tumor specimens. Numbers in parentheses are the number ofpatients when samples were analyzed. Specimens from 2 of 12patients included either tumor or peritoneum.*Denotes significantly different from tumor: P = 0.004.cDenotes significantly different from tumor: P = 0.003.





PGES-1 and PGES-2 were each coexpressed on CD163+ MO/MA in the peritoneum of EOC patients (Fig. 3) althoughcostaining for COX-1 and PGES-1 seemed more prominent.15-LOX2, an enzyme responsible for 15-HETE production wascoexpressed on tumor cells (Fig. 2) and on surface mesothelialcells, stromal cells, on a few of the resident MO/MA, and in thestroma of benign and EOC patients (Figs. 3 and 4). Similarly,5-LOX, an enzyme responsible for synthesis of 5-HETE andwhich contributes to the downstream leukotriene pathway, wascoexpressed on tumor cells and MO/MA (data not shown).

Eicosanoid pathway gene profiling. There were 36 patientswith ovarian or Mullerian-type malignancies and 15 with benignovarian or uterine pathology who provided specimens forgene profiling. The 36 patients in the malignant group includedthe following demographics: median age, 65 years (range,34-80); histopathology, serous (22), serous/endometrioid, endo-metrioid (4 each), undifferentiated (3) mucinous (2), andmixedmalignant Mullerian tumor (1); stage I (1), stage II (4), stage III(23), stage IV (8). Twenty-seven of 35 samples had tumor andMP specimens from the samepatients (one patient hadMPonly).

Fig. 2. Frozen sections of serous and endometrioidcancer tissue from EOC patient ID297 wasdouble-stained with epithelial cytokeratin (CK) andeicosanoid pathway components. Overlay imagesof patient with EOC showed colocalization ofcytokeratin (blue) with eicosanoid pathwaycomponents (red) appearing magenta on costainedcells. Colocalization studies showed tumor cellsstrongly positive for COX-1and PGES-1, but weaklypositive or absent costaining for COX-2 andPGES-2. Costaining for15-LOX2 showed that someparts of the tumor were positive (magnification,�400). H&E-stained section shown for comparison.





The 15 patients in the benign group included: median age,53 years (range, 28-82); histopathology, chronic pelvic inflam-matory disease (PID) (two hydrosalpinges, one chronicsalpingitis), three; cystadenofibroma (ovary), fibrothecoma(including two leiomyomatas), two each; cystadenofibroma(ovary and appendix), corpus luteum, fibromatous nodule (andleiomyoma), adenomyosis, cystic teratoma (and serous adeno-fibroma), mucinous tumor, low malignant potential (LMP)and endometriosis (and LMP/serous cystadenofibroma), oneeach. Genes differentially expressed (P < 0.05) among MP, BP,and tumor are shown in Table 2 and Fig. 5. Ratios are shownaccording to the central method for display using a normaliza-tion factor (20).Based on our previous finding that sPLA2 was differentially

expressed in MP compared with tumor, we extended the

comparison of tumor with MP as well as BP using anexpanded list of selected genes associated with eicosanoidmetabolites and arachidonic acid metabolism pathways. Theresults show that of the 49 genes that were analyzed, 17genes were differentially expressed in MP compared withtumor and 15 genes in BP compared with tumor (Table 2;Fig. 5), the exception being cytochrome P450, family 2,subfamily J, polypeptide 2 (CYP2J2), also called arachidonicacid epoxygenase, which was increased in tumor relative toMP and BP. No multiple comparison correction analysiswas done.The list of genes that we found increased in BP or MP relative

to tumor tissue include the following: AKRIC3 (catalyzes thereduction of prostaglandins—formation of 9a11hPGE2 fromPGD2, PGH2, in the presence of NADPH); CBR3 (carbonyl

Fig. 3. Peritoneal tissue from a patient with EOC (patient no. 294) costained for the macrophage marker, CD163, and eicosanoid pathway components. Overlay images ofEOC patient showed colocalization of CD163 (green) and the eicosanoid pathway enzymes: COX-1, COX-2, PGES-1, PGES-2,15-LOX2 (red) producing a yellow stain(magnification, �400). H&E-stained sections are shown for comparison.





reductase or PGE2 9 reductase)—metabolizes prostaglandins,steroids, and various pharmacologic agents; glutathiamineperoxidase (GPX3) inhibits 5-LOX—catalyzed with H2O2 andlipid hydroperoxide; LYPLA3—regulates phospholipids lyso-zymal enzyme and has Ca-independent PLA2 and transcyclaseactivity; FBXl7 involved with phosphorylation-dependentubiquitination. In addition to the above enzymes, a numberof PLA2s and a lysozymal phospholipases were increased in MPand often in BP tissues compared with tumor tissue (Table 2).In contradistinction to these genes, CYP2J2 , arachidonic acidepoxygenase which catalyzes the reactions involved with drugmetabolism, cholesterol, and other lipids, was decreased in MPand BP relative to tumor.

Discussion

Our data show that PGE2 concentrations were increased intumor tissue compared with MP or BP, as was a grouping ofPGE2 and three other eicosanoids having the potential to driveproliferation of tumor and/or increase capillary permeability.The elevated eicosanoids within EOC tissue specimens seemsconsistent with the expression pattern seen for COX-1 andPGES-1, versus COX-2 and PGES-2, which are only poorlyexpressed. The latter is also in agreement with recent reportsshowing that COX-1 but not COX-2 was enhanced at both theRNA and protein levels in EOC (19), and that PGE2 seems to beregulated by COX-1 but not by COX-2 (21). Vascular

Fig. 4. Photomicrographs from the peritoneal tissueof patient no. 283 with benign cystic teratoma ofovary which was double-stained with antibodies tomacrophage marker, CD163, and eicosanoid pathwaycomponents. Surface mesothelial cells were stronglypositive for15-LOX2, COX-1, and PGES-1, but weakPGES-2 staining, and absent expression of COX-2.Some resident MO/MA costain for COX-1, PGES-1, and15-LOX (magnification, �400). H&E-stained section isshown for comparison.





endothelial growth factor, a promoter of angiogenesis in EOC,is also selectively inhibited by a COX-1 inhibitor—an effectreversed by PGE2 (19).Using differential gene expression analysis, we showed that a

number of genes involved with bioactive lipids within MP orBP specimens were up-regulated when compared with the geneexpression within EOC tumor specimens. These genes arespecifically linked to eicosanoid and arachidonic acid metab-olism pathways. These findings seemed to contrast with ourresults showing elevated tissue levels of PGE2 and other

eicosanoids compared with MP or BP in the tissue. However,several of the genes, such as AKR1C3, CBR3, GPX3, andLYPLA3 , may actually serve to regulate levels of prominenteicosanoids. Moreover, 15-hydroxyprostaglandin dehydroxyge-nase was recently shown to contribute to the inactivation anddegradation of prostaglandins in colon cancer (22), supportinga tumor suppressor role for this enzyme.Increased phospholipase activity observed in MP and BP

would likely represent an early step for the production ofeicosanoids including PGE2, through the release of arachidonic

Table 2. Differential analysis of genes involved with eicosanoid metabolism in BP, MP, and tumor tissue fromEOC patients

Gene symbol Gene name Two-sample t test(P < 0.05)

ParametricP value

PermutationP value

BP/MP MP/TU BP/TU BP/MP/TU (10,000)

AKR1C3 Aldo-keto reductase family 1, member C3 0.7193 0.0002 0.0090 0.0003 0.000ALOX12 Arachidonate 12-LOX 0.7210 0.1933 0.1372 0.2139 0.219ALOX15B Arachidonate 15-LOX, type B 0.0845 0.3204 0.4056 0.2809 0.282ALOX5 Arachidonate 5-LOX 0.2851 0.2413 0.9839 0.3956 0.394ALOX5AP Arachidonate 5-LOX-activating protein 0.4968 0.2594 0.1621 0.2807 0.274CBR1 Carbonyl reductase 1 0.9521 0.6500 0.6845 0.8725 0.865CBR3 Carbonyl reductase 3 0.3450 0.0752 0.0114 0.0356 0.035COX7A1 Cytochrome c oxidase subunit VIIa polypeptide 1, muscle 0.4768 0.0000 0.0015 0.0000 0.000CYP2B6 Cytochrome P450, family 2, subfamily B, polypeptide 6 0.5770 0.5552 0.3043 0.5800 0.574CYP2C8 Cytochrome P450, family 2, subfamily C, polypeptide 8 0.8174 0.1594 0.1248 0.2096 0.211CYP2C9 Cytochrome P450, family 2, subfamily C, polypeptide 9 0.5779 0.8413 0.6053 0.7828 0.791CYP2E1 Cytochrome P450, family 2, subfamily E, polypeptide 1 0.2687 0.3136 0.8311 0.4099 0.416CYP2J2 Cytochrome P450, family 2, subfamily J, polypeptide 2 0.0004 0.0023 0.0000 0.0000 0.000CYP4A11 Cytochrome P450, family 4, subfamily A, polypeptide 11 0.9058 0.9466 0.8511 N/A N/ACYP4F2 Cytochrome P450, family 4, subfamily F, polypeptide 2 0.0727 0.0995 0.4900 0.0916 0.092CYP4F3 Cytochrome P450, family 4, subfamily F, polypeptide 3 0.5136 0.3154 0.8870 0.6193 0.627EPHX1 Epoxide hydrolase 1, microsomal (xenobiotic) 0.8415 0.3871 0.3502 0.5559 0.553EPHX2 Epoxide hydrolase 2, cytoplasmic 0.5289 0.9173 0.4591 0.7285 0.741FBXL7 F-box and leucine-rich repeat protein 7 0.7134 0.0000 0.0025 0.0000 0.000GGT1 g-Glutamyltransferase 1 0.6476 0.0565 0.3592 0.2129 0.215GGTLA1 g-Glutamyltransferase-like activity 1 0.5308 0.7916 0.4056 0.7154 0.727GPX1 Glutathione peroxidase 1 0.4529 0.9342 0.5333 0.7624 0.766GPX2 Glutathione peroxidase 2 (gastrointestinal) 0.4564 0.6154 0.2140 0.5265 0.524GPX3 Glutathione peroxidase 3 (plasma) 0.0796 0.0035 0.0000 0.0001 0.000GPX4 Glutathione peroxidase 4 (phospholipid hydroperoxidase) 0.9954 0.0607 0.2367 0.1554 0.154LTA4H Leukotriene A4 hydrolase 0.7920 0.8280 0.9303 N/A N/ALTC4S Leukotriene C4 synthase 0.0369 0.2078 0.3529 0.1536 0.154LYPLA3 Lysophospholipase 3 (lysosomal PLA2) 0.0033 0.0050 0.8711 0.0071 0.006PGDS Prostaglandin D2 synthase, hematopoietic 0.2509 0.0024 0.1265 0.0074 0.007PLA2G12A PLA2, group XIIA 0.0863 0.3368 0.4769 0.4018 0.396PLA2G1B PLA2, group IB (pancreas) 0.2182 0.5993 0.3238 0.2683 0.264PLA2G2A PLA2, group IIA (platelets, synovial fluid) 0.0800 0.0000 0.0026 0.0000 0.000PLA2G4A PLA2, group IVA 0.2309 0.1360 0.9387 0.2604 0.267PLA2G4C PLA2, group IVC 0.1512 0.0001 0.0000 0.0000 0.000PLA2G5 PLA2, group V 0.9747 0.0557 0.0791 0.1179 0.117PLA2G6 PLA2, group VI 0.2847 0.0000 0.0001 0.0000 0.000PLA2R1 PLA2 receptor 1, 180 kDa 0.6062 0.0002 0.0143 0.0006 0.001PLCB1 Phospholipase C, h1 (phosphoinositide-specific) 0.5588 0.5584 0.9221 0.7829 0.777PPARG Peroxisome proliferative-activated receptor, g 0.9174 0.0001 0.0009 0.0000 0.000PTGER2 Prostaglandin E receptor 2 (subtype EP2), 53 kDa 0.8915 0.2444 0.3140 0.4484 0.457PTGER3 Prostaglandin E receptor 3 (subtype EP3) 0.4510 0.0000 0.0014 0.0000 0.000PTGER4 Prostaglandin E receptor 4 (subtype EP4) 0.7645 0.0815 0.2483 0.2206 0.223PTGES Prostaglandin E synthase 0.6377 0.8419 0.7255 0.8771 0.883PTGFR Prostaglandin F receptor (FP) 0.6346 0.0000 0.0000 0.0000 0.000PTGIR Prostaglandin I2 (prostacyclin) receptor (IP) 0.3518 0.0157 0.0039 0.0048 0.006PTGIS Prostaglandin I2 (prostacyclin) synthase 0.4689 0.0001 0.0094 0.0002 0.000PTGS2 Prostaglandin-endoperoxide synthase 2 0.9421 0.0993 0.1180 0.1521 0.154TBXA2R Thromboxane A2 receptor 0.7927 0.9388 0.7286 N/A N/ATBXAS1 Thromboxane A synthase 1 0.8030 0.0345 0.0881 0.0760 0.078

NOTE: P values showing significant differential down-regulation (italics) and up-regulation (boldface).





acid from membranes. The presence and relatively higherexpression of enzyme transcripts involved with inhibition ordegradation alongside production, which suggests that produc-tion might be coordinately regulated in normal tissues, or evenMP, is intriguing. Conversely, the loss of tight regulation intumor tissue may be an etiologic factor in this disease.Present data may implicate inflammation, perhaps sustained

and produced by certain eicosanoids as potentially importantin the development and progression of EOC. The role of otherbioactive lipids typically associated with inhibition of prolifer-

ation also requires further examination. The tumor suppressorfunctions of 15-LOX2, for example, were previously shown in aprostate model (23). Here, we show that the peritoneum ofnormal subjects sometimes expressed 15-LOX2 and its product15-HETE. These findings could suggest a protective role for15-HETE in the absence of elevated levels of PGE2 in thenormal peritoneum (22). Collectively, our data suggest that thepresence of elevated levels of certain eicosanoids such as PGE2,12-HETE, 5-HETE, and perhaps LTB4, might promote tumorprogression, whereas others, such as 15-HETE and 13-HODE,might interfere with the progression to malignancy. Thisconcept has also been considered by others (24) but not inEOC.A number of genes that were differentially expressed in MP,

BP, or tumor have previously been associated with cancer, if notspecifically with EOC itself. For example, CYP2J2 , which wasdifferentially expressed in MP or BP, converts arachidonic acidto four regioisomeric epoxyeicosatrienoic acids. Although theexact biological role of epoxyeicosatrienoics is unclear, a recentreport (25) has shown a strong presence of epoxyeicosatrie-noics in human carcinoma, but not in normal tissues. This wasaccompanied by the activation of mitogen-activated proteinkinases and phosphoinositide-3-kinase/Akt systems as well asthe elevation of epithelial growth factor receptor phosphoryla-tion, all of which suggest a role in promoting a neoplasticcellular phenotype.Prostacyclin synthase (PTGIS) was differentially increased in

MP or BP versus tumor. Inactivation of specific tumorsuppressor genes by transcriptional silencing associated withhypermethylation of the promoter is common in cancer. Usingreverse transcription-PCR, Frigola et al. (26) have shown thatPTGIS was inactivated through hypermethylation of itspromoter region. Prostacyclin seems to exhibit both antiproli-ferative effects (27) and chemopreventive properties (28).Down-regulation of the enzyme responsible for prostacyclinsynthesis would contribute to loss of this important prostanoidcompound at the tumor site.Overexpression of prostaglandin D synthase in EOC has

recently been reported (29), although we found prostaglandinD synthase to be differentially expressed in MP versus tumor.Expression of prostaglandin D synthase mRNA was found intumor cells of all various types of EOC and relative stainingintensity seemed to be selective for certain types of disease.Thromboxane synthase metabolizes the cyclooxygenase

product, prostaglandin H (2), into thromboxane A (2).Thromboxane synthase has been found to be weakly expressedor absent in normal differentiated or advanced prostate tumors,and markedly increased in tumors with perineural invasion(30, 31), and in adenocarcinoma and squamous cell carcinomaof the lung (32). The relative expression of thromboxanesynthase in normal and tumor peritoneum from patients withcancer has not previously been reported but its overexpressionin this disease might be a poor prognostic factor.Prostaglandins typically require specific receptors to bring

about their pharmacodynamic actions. It is interesting to notethat prostaglandin E receptor gene, also differentially expressedinMPor BP versus tumor, is up-regulated by PGE2 (33). Althoughthe consequence of increased gene expression in EOC is unclear,PGE2 stimulation of the prostaglandin E3 receptor in lungadenocarcinoma led to a direct increase in Src activity and isbelieved to be a contributing factor in tumor progression (34).

Fig. 5. Selected genes differentially expressed among MP, BP, and tumor tissues(univariate t test, P < 0.05).The identity of each sample tissue type is shown and thesame type of samples labeled under color bars.





Peroxisome proliferator-activated receptor-g is a ligand-activated transcription factor that, in addition to its well-established role in lipid and glucose metabolism, is known tocontrol cell proliferation and differentiation in several tissues.Vignati et al. (35) recently showed that peroxisome proliferator-activated receptor-g was expressed on epithelial ovarian tumortissues but not in normal ovarian tissue. Our data shows higherdifferential expression in MP and BP versus tumor.Because of their importance to the biology of cancer, certain

eicosanoids including their enzymes and receptor targets mightrepresent appropriate specific targets for therapy or diagnosis.New therapeutic strategies might include molecules that alterthe ratio of different eicosanoids such as the ratio of PGE3 toPGE2 (36). Others might selectively block PLA2 enzymes,thereby limiting the release of arachidonic acid from cellsurface membranes (37), or selectively block synthesis or

inhibit production of PGE2, COX-1 and COX-2, 12-HETE, and5-HETE. The studies reported above (19, 21) might also suggesta role for COX-1 inhibition in the control of vascular endo-thelial growth factor.An inflammatory process is clearly a part of the tumor

microenvironment of EOC (9, 10). There is increasing supportfor a linkage between the inflammatory process in EOC andeicosanoid and arachidonic pathways. Using a combination ofelectrospray mass tandem spectroscopy and cDNA microarrayanalysis, we have shown significant differences in the distribu-tion of a number of these elements in the tumor and non–tumor-involved peritoneum and in the peritoneum of patientswith benign disease. The emerging patterns could provide abasis for further studies aimed at establishing critical pathwaysand the strategies that could affect the prevention or treatmentof EOC.



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2007;13:5736-5744. Clin Cancer Res Ralph S. Freedman, Ena Wang, Sonia Voiculescu, et al. and Pathways in Advanced Ovarian CancerComparative Analysis of Peritoneum and Tumor Eicosanoids

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