sign in sign up
<<
Home , CYP2B6, CYP2C8, CYP2C9, CYP2E1, CYP2J2, CYP4A11

Human Biology

Comparative Analysis of Peritoneum and Tumor 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 profile and differentially expressed eicosanoid and pathway 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 E2 (PGE2), the hydroxyeicosate- traenoic acids (12-HETE and 5-HETE), and (LTB4), selected for tumor growth potential, and two other bioactive (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 (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 and (COX) and lipoxygenase (LOX) variously (, , 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- 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 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 , 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.); -1(PGES-1; spectrometry analysis combined with 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 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.). peritoneum samples were obtained from patients with EOC and pelvic Negative controls employed secondary antibodies alone. peritoneum samples from patients with benign pelvic disease and EOC Tissue sections were mounted with Slow-Fade Gold Anti-Fade tumors. All specimens were obtained under an Institutional Review reagent (Molecular Probes) and viewed with an Olympus FV500 laser Board–approved protocol. MP specimens with contaminating tumor as scanning confocal microscope; images were captured at 400Â and identified by a gynecologic oncology pathologist (M. Deavers) were 600Â magnification by Fluoview software Version 4.3. excluded from eicosanoid and microarray analyses. Specimens for Differential gene expression analysis. Specimen collection and eicosanoid analyses were obtained in the operating room, snap-frozen sample preparation were as described previously (9) but with a directly, and stored at -80jC until analyzed. Endogenous eicosanoids substantially increased sample size. Specimens for microarray analyses were measured with a Quattro Ultima tandem mass spectrometer were transferred directly to the laboratory from the operating room in (Micromass) equipped with an Agilent HP1100 binary high-pressure cold saline, then snap-frozen in RNAlate (Ambion, Inc.), stored at liquid chromatography inlet. Eicosanoids were separated on a Luna 5A -80jC. Processing of frozen material and extraction of RNA with quality Phenyl-Hexyl 2 Â 150 mm column (Phenomenex) with a rapid linear controls were previously described (9). Among the specimens, 15 of gradient of 70% to 90% methanol in 4 min over 10 min. The mobile them were from peritoneum associated with benign pelvic disease, phase consisted of 10 mmol/L of ammonium (pH 8.5) and designated ‘‘BP’’, 35 specimens from EOC or histopathologically related methanol (30:70). Fragmentation of all compounds was achieved by tumors, designated ‘‘Tu’’, and 27 tumor-free peritoneal specimens using argon as the collision gas. This method produces excellent designated ‘‘MP.’’ Specimens that had microscopic tumor invasion in linearity and a lower limit of quantification for eicosanoids of 1ng/mL, subsequent histopathologic examination were excluded. Total RNA which is adequate to assess endogenous eicosanoid metabolism that isolation, RNA amplification and array hybridization were done as commonly occurred as ng/mg of protein in 5 Â 106 cell aliquots or described (18). Custom microarrays were printed at the Immunoge- tissue samples. We have used this method to characterize in detail netics Section, Department of Transfusion Medicine, Clinical Center, eicosanoid metabolism in human lung, prostate, breast, and colon NIH with a configuration of 32 Â 24 Â 23 and containing 17,500 tissues as well as in numerous cell lines. The following eicosanoids were elements. For a complete list of genes included in the Hs-CCDTM- 7 measured: PGE2 (product of pathway), 5-HETE 17.5k-1px, printing is available at our web site. Genes involved in (product of 5-lipoxygenase or 5-LOX), LTB4 (product of leukotrienes- eicosanoid and arachidonic acid pathways from the cDNA microarray A4 and downstream product of 5-LOX), 12-HETE (product of data set were selected according to the KEGG pathway finder8 from the 12-LOX), 15-HETE (product of 15-LOX2), and 13-HODE (product of

7 http://nciarray.nci.nih.gov/gal_files/index.shtml 6 R.A. Newman, P. Yang, unpublished data. 8 http://www.genome.jp/kegg/pathway.html

www.aacrjournals.org 5737 Clin Cancer Res 2007;13(19) October 1,2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology

patients with EOC. Eleven of 12 patients in the malignant group had stage III or IV carcinomas with serous components. Patients with benign pathology included entities such as benign ovarian epithelial or germ cell tumors (two patients), low malignant potential tumors of the ovary without invasive implants (four patients), chronic pelvic inflammatory disease (two patients). Median ages were 68 (range, 61-80) for the malignant group and 62 (range, 28-78) for the benign group. There were eight matched specimens of tumor and MP peritoneum and separate MP or tumor specimens from four patients, and BP was obtained from eight patients with a variety of benign pelvic conditions who were scheduled for pelvic abdominal surgery. Only MP specimens that were tumor-free were 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 tissue protein. PGE2, an eicosanoid commonly associated with malignant 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) are averaged, they show a significant linear trend upward from BP through 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 move in the opposite direction, the trends were not statistically significant (Fig. 1; Table 1). Immunocostaining of eicosanoid pathway enzymes. We next examined coexpression of eicosanoid products by IIF using confocal microscopy on: tumor tissue, peritoneum of EOC patients, and peritoneum from patients with benign pelvic disease on two to three individual patients. We showed, as others have (19), that COX-1 expression was strongly expressed on the tumor cells, whereas COX-2 was only weakly expressed on tumor cell islets. In addition, expression of PGES-1and PGES-2 paralleled the results for COX-1and COX-2 with weak expression of PGES-2 (Fig. 2). In contrast, COX-1, COX-2, and

Fig. 1. Boxplots of median, quartiles, and range for level of eicosanoids PGE2, LTB4, 15-HETE, 12-HETE, 5-HETE, and 13-HODE in BP, MP, and tumor tissues. A, individual eicosanoid values; B, average combined PGE2,LTB4, 12-HETE, and Table 1. Overall and individual tissue comparison 5-HETE values. of eicosanoid values in BP, MP, and tumor tissues complete cDNA microarray data set after normalization and back- Variables Comparison of mean 9 ground subtraction, using BRB array tool. Two-sample t tests were eicosanoid values (ng/mg) done between MP versus BP, MP versus tumor and BP versus tumor. In BP (N)MP(N) Tumor addition, F tests (n = 3) were also done with 10,000 random c permutations using the BRB array tool. Genes with P < 0.05 were PGE2 1.14 (8)* 1.60 (10) 6.86 (10) defined as significantly differentially expressed genes and were 5-HETE 0.56(8) 1.10 (10) 2.07 (10) visualized by cluster and TreeView analysis. A selected gene group was 12-HETE 1.95 (8) 4.37 (10) 5.60 (10) LTB 0.57 (8) 1.68 (10) 0.86 (10) used in the analysis. 4 15-HETE 16.05 (8) 6.55 (10) 6.85 (10) 13-HODE 4.62 (8) 4.88 (10) 2.52 (10) Results

Eicosanoid metabolites increased in EOC tumor, MP, and BP NOTE: Mean values were from eight pairs of BP samples and from one to two samples/patients obtained from 10 MP and from 10 tissues. Eicosanoid metabolite levels were measured on frozen tumor specimens. Numbers in parentheses are the number of tumor specimens obtained from 12 -naBve patients when samples were analyzed. Specimens from 2 of 12 patients included either tumor or peritoneum. *Denotes significantly different from tumor: P = 0.004. cDenotes significantly different from tumor: P = 0.003. 9 http://linus.nci.nih.gov/BRB-ArrayTools.html

Clin Cancer Res 2007;13(19) October 1, 2007 5738 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

Fig. 2. Frozen sections of serous and endometrioid cancer tissue from EOC patient ID297 was double-stained with epithelial cytokeratin (CK)and eicosanoid pathway components. Overlay images of patient with EOC showed colocalization of cytokeratin (blue) with eicosanoid pathway components (red) appearing magenta on costained cells. Colocalization studies showed tumor cells strongly positive for COX-1and PGES-1, but weakly positive or absent costaining for COX-2and PGES-2. Costaining for 15-LOX2 showed that some parts of the tumor were positive (magnification, Â400). H&E-stained section shown for comparison.

PGES-1and PGES-2 were each coexpressed on CD163+MO/ Eicosanoid pathway gene profiling. There were 36 patients MA in the peritoneum of EOC patients (Fig. 3) although with ovarian or Mullerian-type malignancies and 15 with benign costaining for COX-1and PGES-1seemed more prominent. ovarian or uterine pathology who provided specimens for 15-LOX2, an responsible for 15-HETE production was gene profiling. The 36 patients in the malignant group included coexpressed on tumor cells (Fig. 2) and on surface mesothelial the following demographics: median age, 65 years (range, cells, stromal cells, on a few of the resident MO/MA, and in the 34-80); histopathology, serous (22), serous/endometrioid, endo- stroma of benign and EOC patients (Figs. 3 and 4). Similarly, metrioid (4 each), undifferentiated (3) mucinous (2), and mixed 5-LOX, an enzyme responsible for synthesis of 5-HETE and malignant Mullerian tumor (1); stage I (1), stage II (4), stage III which contributes to the downstream leukotriene pathway, was (23), stage IV (8). Twenty-seven of 35 samples had tumor and coexpressed on tumor cells and MO/MA (data not shown). MP specimens from the same patients (one patient had MP only).

www.aacrjournals.org 5739 Clin Cancer Res 2007;13(19) October 1,2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology

The 15 patients in the benign group included: median age, comparison of tumor with MP as well as BP using an 53 years (range, 28-82); histopathology, chronic pelvic inflam- expanded list of selected genes associated with eicosanoid matory disease (PID) (two hydrosalpinges, one chronic metabolites and arachidonic acid metabolism pathways. The salpingitis), three; cystadenofibroma (ovary), fibrothecoma results show that of the 49 genes that were analyzed, 17 (including two leiomyomatas), two each; cystadenofibroma genes were differentially expressed in MP compared with (ovary and appendix), corpus luteum, fibromatous nodule (and tumor and 15 genes in BP compared with tumor (Table 2; leiomyoma), adenomyosis, cystic teratoma (and serous adeno- Fig. 5), the exception being P450, family 2, fibroma), mucinous tumor, low malignant potential (LMP) subfamily J, polypeptide 2 (CYP2J2), also called arachidonic and endometriosis (and LMP/serous cystadenofibroma), one acid , which was increased in tumor relative to each. Genes differentially expressed (P < 0.05) among MP, BP, MP and BP. No multiple comparison correction analysis and tumor are shown in Table 2 and Fig. 5. Ratios are shown was done. according to the central method for display using a normaliza- The list of genes that we found increased in BP or MP relative tion factor (20). to tumor tissue include the following: AKRIC3 (catalyzes the Based on our previous finding that sPLA2 was differentially reduction of prostaglandins—formation of 9a11hPGE2 from expressed in MP compared with tumor, we extended the PGD2, 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 of EOC 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, Â400). H&E-stained sections are shown for comparison.

Clin Cancer Res 2007;13(19) October 1, 2007 5740 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

Fig. 4. Photomicrographs from the peritoneal tissue of patient no. 283 with benign cystic teratoma of ovary which was double-stained with antibodies to macrophage marker, CD163, and eicosanoid pathway components. Surface mesothelial cells were strongly positive for 15-LOX2, COX-1, and PGES-1, but weak PGES-2staining, and absent expression of COX-2. Some resident MO/MA costain for COX-1, PGES-1, and 15-LOX (magnification, Â400). H&E-stained section is shown for comparison.

reductase or PGE2 9 reductase)—metabolizes prostaglandins, Discussion , and various pharmacologic agents; glutathiamine Our data show that PGE concentrations were increased in peroxidase (GPX3) inhibits 5-LOX—catalyzed with H2O2 and 2 hydroperoxide; LYPLA3—regulates phospholipids lyso- tumor tissue compared with MP or BP, as was a grouping of PGE and three other eicosanoids having the potential to drive zymal enzyme and has Ca-independent PLA2 and transcyclase 2 activity; FBXl7 involved with phosphorylation-dependent proliferation of tumor and/or increase capillary permeability. ubiquitination. In addition to the above enzymes, a number The elevated eicosanoids within EOC tissue specimens seems of PLA2s and a lysozymal phospholipases were increased in MP consistent with the expression pattern seen for COX-1and and often in BP tissues compared with tumor tissue (Table 2). PGES-1, versus COX-2 and PGES-2, which are only poorly In contradistinction to these genes, CYP2J2, arachidonic acid expressed. The latter is also in agreement with recent reports epoxygenase which catalyzes the reactions involved with drug showing that COX-1but not COX-2 was enhanced at both the metabolism, , and other lipids, was decreased in MP RNA and protein levels in EOC (19), and that PGE2 seems to be and BP relative to tumor. regulated by COX-1but not by COX-2 (21).Vascular

www.aacrjournals.org 5741 Clin Cancer Res 2007;13(19) October 1,2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology endothelial growth factor, a promoter of in EOC, eicosanoids compared with MP or BP in the tissue. However, is also selectively inhibited by a COX-1inhibitor—an effect several of the genes, such as AKR1C3, CBR3, GPX3, and LYPLA3 reversed by PGE2 (19). , may actually serve to regulate levels of prominent Using differential gene expression analysis, we showed that a eicosanoids. Moreover, 15-hydroxyprostaglandin dehydroxyge- number of genes involved with bioactive lipids within MP or nase was recently shown to contribute to the inactivation and BP specimens were up-regulated when compared with the gene degradation of prostaglandins in colon cancer (22), supporting expression within EOC tumor specimens. These genes are a tumor suppressor role for this enzyme. specifically linked to eicosanoid and arachidonic acid metab- Increased phospholipase activity observed in MP and BP olism pathways. These findings seemed to contrast with our would likely represent an early step for the production of results showing elevated tissue levels of PGE2 and other eicosanoids including PGE2, through the release of arachidonic

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

Gene symbol Gene name Two-sample t test Parametric Permutation (P < 0.05) P value P 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 0.000 ALOX12 Arachidonate 12-LOX 0.7210 0.1933 0.1372 0.2139 0.219 ALOX15B Arachidonate 15-LOX, type B 0.0845 0.3204 0.40560.2809 0.282 ALOX5 Arachidonate 5-LOX 0.2851 0.2413 0.9839 0.39560.394 ALOX5AP Arachidonate 5-LOX-activating protein 0.4968 0.2594 0.1621 0.2807 0.274 CBR1 Carbonyl reductase 1 0.9521 0.6500 0.6845 0.8725 0.865 CBR3 Carbonyl reductase 3 0.3450 0.0752 0.0114 0.0356 0.035 COX7A1 Cytochrome c oxidase subunit VIIa polypeptide 1, muscle 0.4768 0.0000 0.0015 0.0000 0.000 CYP2B6 , family 2, subfamily B, polypeptide 60.5770 0.5552 0.3043 0.5800 0.574 CYP2C8 Cytochrome P450, family 2, subfamily C, polypeptide 8 0.8174 0.1594 0.1248 0.20960.211 CYP2C9 Cytochrome P450, family 2, subfamily C, polypeptide 9 0.5779 0.8413 0.6053 0.7828 0.791 CYP2E1 Cytochrome P450, family 2, subfamily E, polypeptide 1 0.2687 0.3136 0.8311 0.4099 0.416 CYP2J2 Cytochrome P450, family 2, subfamily J, polypeptide 2 0.0004 0.0023 0.0000 0.0000 0.000 CYP4A11 Cytochrome P450, family 4, subfamily A, polypeptide 11 0.9058 0.9466 0.8511 N/A N/A CYP4F2 Cytochrome P450, family 4, subfamily F, polypeptide 2 0.0727 0.0995 0.4900 0.09160.092 CYP4F3 Cytochrome P450, family 4, subfamily F, polypeptide 3 0.5136 0.3154 0.8870 0.6193 0.627 EPHX1 hydrolase 1, microsomal (xenobiotic) 0.8415 0.3871 0.3502 0.5559 0.553 EPHX2 2, cytoplasmic 0.5289 0.9173 0.4591 0.7285 0.741 FBXL7 F-box and -rich repeat protein 7 0.7134 0.0000 0.0025 0.0000 0.000 GGT1 g-Glutamyltransferase 1 0.6476 0.0565 0.3592 0.2129 0.215 GGTLA1 g-Glutamyltransferase-like activity 1 0.5308 0.79160.4056 0.7154 0.727 GPX1 peroxidase 1 0.4529 0.9342 0.5333 0.7624 0.766 GPX2 Glutathione peroxidase 2 (gastrointestinal) 0.4564 0.6154 0.2140 0.5265 0.524 GPX3 Glutathione peroxidase 3 (plasma) 0.0796 0.0035 0.0000 0.0001 0.000 GPX4 Glutathione peroxidase 4 (phospholipid hydroperoxidase) 0.9954 0.0607 0.2367 0.1554 0.154 LTA4H hydrolase 0.7920 0.8280 0.9303 N/A N/A LTC4S synthase 0.0369 0.2078 0.3529 0.15360.154

LYPLA3 Lysophospholipase 3 (lysosomal PLA2) 0.0033 0.0050 0.8711 0.0071 0.006 PGDS synthase, hematopoietic 0.2509 0.0024 0.1265 0.0074 0.007

PLA2G12A PLA2, group XIIA 0.0863 0.3368 0.4769 0.4018 0.396 PLA2G1B PLA2, group IB (pancreas) 0.2182 0.5993 0.3238 0.2683 0.264 PLA2G2A PLA2, group IIA (, synovial fluid) 0.0800 0.0000 0.0026 0.0000 0.000 PLA2G4A PLA2, group IVA 0.2309 0.1360 0.9387 0.2604 0.267 PLA2G4C PLA2, group IVC 0.1512 0.0001 0.0000 0.0000 0.000 PLA2G5 PLA2, group V 0.9747 0.0557 0.0791 0.1179 0.117 PLA2G6 PLA2, group VI 0.2847 0.0000 0.0001 0.0000 0.000 PLA2R1 PLA2 1, 180 kDa 0.6062 0.0002 0.0143 0.0006 0.001 PLCB1 , h1 (phosphoinositide-specific) 0.5588 0.5584 0.9221 0.7829 0.777 PPARG proliferative-activated receptor, g 0.9174 0.0001 0.0009 0.0000 0.000 PTGER2 Prostaglandin E receptor 2 (subtype EP2), 53 kDa 0.8915 0.2444 0.3140 0.4484 0.457 PTGER3 Prostaglandin E receptor 3 (subtype EP3) 0.4510 0.0000 0.0014 0.0000 0.000 PTGER4 Prostaglandin E receptor 4 (subtype EP4) 0.7645 0.0815 0.2483 0.2206 0.223 PTGES Prostaglandin E synthase 0.6377 0.8419 0.7255 0.8771 0.883 PTGFR Prostaglandin F receptor (FP) 0.6346 0.0000 0.0000 0.0000 0.000 PTGIR Prostaglandin I2 () receptor (IP) 0.3518 0.0157 0.0039 0.0048 0.006 PTGIS Prostaglandin I2 (prostacyclin) synthase 0.4689 0.0001 0.0094 0.0002 0.000 PTGS2 Prostaglandin-endoperoxide synthase 2 0.9421 0.0993 0.1180 0.1521 0.154 TBXA2R A2 receptor 0.7927 0.9388 0.7286N/A N/A TBXAS1 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).

Clin Cancer Res 2007;13(19) October 1, 2007 5742 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

ation also requires further examination. The tumor suppressor functions of 15-LOX2, for example, were previously shown in a prostate model (23). Here, we show that the peritoneum of normal subjects sometimes expressed 15-LOX2 and its product 15-HETE. These findings could suggest a protective role for 15-HETE in the absence of elevated levels of PGE2 in the normal peritoneum (22). Collectively, our data suggest that the presence of elevated levels of certain eicosanoids such as PGE2, 12-HETE, 5-HETE, and perhaps LTB4, might promote tumor progression, whereas others, such as 15-HETE and 13-HODE, might interfere with the progression to malignancy. This concept has also been considered by others (24) but not in EOC. A number of genes that were differentially expressed in MP, BP, or tumor have previously been associated with cancer, if not specifically with EOC itself. For example, CYP2J2, which was differentially expressed in MP or BP, converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids. Although the exact biological role of epoxyeicosatrienoics is unclear, a recent report (25) has shown a strong presence of epoxyeicosatrie- noics in human carcinoma, but not in normal tissues. This was accompanied by the of mitogen-activated protein kinases and phosphoinositide-3-kinase/Akt systems as well as the elevation of epithelial growth factor receptor phosphoryla- tion, all of which suggest a role in promoting a neoplastic cellular phenotype. (PTGIS) was differentially increased in MP or BP versus tumor. Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is common in cancer. Using reverse transcription-PCR, Frigola et al. (26) have shown that PTGIS was inactivated through hypermethylation of its promoter region. Prostacyclin seems to exhibit both antiproli- ferative effects (27) and chemopreventive properties (28). Down-regulation of the enzyme responsible for prostacyclin synthesis would contribute to loss of this important compound at the tumor site. Overexpression of prostaglandin D synthase in EOC has recently been reported (29), although we found prostaglandin D synthase to be differentially expressed in MP versus tumor. Expression of prostaglandin D synthase mRNA was found in tumor cells of all various types of EOC and relative staining intensity 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 expressed or absent in normal differentiated or advanced prostate tumors, Fig. 5. Selected genes differentially expressed among MP, BP, and tumor tissues and markedly increased in tumors with perineural invasion (univariate t test, P < 0.05).The identity of each sample tissue type is shown and the (30, 31), and in adenocarcinoma and squamous cell carcinoma same type of samples labeled under color bars. of the lung (32). The relative expression of thromboxane synthase in normal and tumor peritoneum from patients with acid from membranes. The presence and relatively higher cancer has not previously been reported but its overexpression expression of enzyme transcripts involved with inhibition or in this disease might be a poor prognostic factor. degradation alongside production, which suggests that produc- Prostaglandins typically require specific receptors to bring tion might be coordinately regulated in normal tissues, or even about their pharmacodynamic actions. It is interesting to note MP, is intriguing. Conversely, the loss of tight regulation in that prostaglandin E receptor gene, also differentially expressed tumor tissue may be an etiologic factor in this disease. in MP or BP versus tumor, is up-regulated by PGE2 (33). Although Present data may implicate , perhaps sustained the consequence of increased gene expression in EOC is unclear, and produced by certain eicosanoids as potentially important PGE2 stimulation of the prostaglandin E3 receptor in lung in the development and progression of EOC. The role of other adenocarcinoma led to a direct increase in Src activity and is bioactive lipids typically associated with inhibition of prolifer- believed to be a contributing factor in tumor progression (34).

www.aacrjournals.org 5743 Clin Cancer Res 2007;13(19) October 1,2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology

Peroxisome proliferator-activated receptor-g is a - inhibit production of PGE2, COX-1and COX-2, 12-HETE,and activated transcription factor that, in addition to its well- 5-HETE. The studies reported above (19, 21) might also suggest established role in lipid and glucose metabolism, is known to a role for COX-1inhibition in the control of vascular endo- control cell proliferation and differentiation in several tissues. thelial growth factor. Vignati et al. (35) recently showed that peroxisome proliferator- An inflammatory process is clearly a part of the tumor activated receptor-g was expressed on epithelial ovarian tumor microenvironment of EOC (9, 10). There is increasing support tissues but not in normal ovarian tissue. Our data shows higher for a linkage between the inflammatory process in EOC and differential expression in MP and BP versus tumor. eicosanoid and arachidonic pathways. Using a combination of Because of their importance to the biology of cancer, certain electrospray mass tandem spectroscopy and cDNA microarray eicosanoids including their enzymes and receptor targets might analysis, we have shown significant differences in the distribu- represent appropriate specific targets for therapy or diagnosis. tion of a number of these elements in the tumor and non– New therapeutic strategies might include molecules that alter tumor-involved peritoneum and in the peritoneum of patients the ratio of different eicosanoids such as the ratio of PGE3 to with benign disease. The emerging patterns could provide a PGE2 (36). Others might selectively block PLA2 enzymes, basis for further studies aimed at establishing critical pathways thereby limiting the release of arachidonic acid from cell and the strategies that could affect the prevention or treatment surface membranes (37), or selectively block synthesis or of EOC.

References 1. Funk CD. Prostaglandins and leukotrienes: advances lipoxygenase-1 in colorectal epithelial cell terminal carcinoma cells and is up-regulated in human tumors. in eicosanoid biology. Science 2001;294:1871 ^ 5. differentiation and tumorigenesis. Cancer Res 2005; Cancer Res 2005;65:4704 ^ 15. 2. Leslie C. Regulation of arachidonic acid availability 65:11492. 26. Frigola J, Munoz M, Clark SJ, MorenoV, Capella G, for eicosanoid production. Biochem Cell Biol 2004; 15. Bhatia B, Tang S, Yang P, et al. Cell-autonomous Peinado MA. Hypermethylation of the prostacyclin 82:1 ^ 17. induction of functional tumor suppressor15-lipoxyge- synthase (PTGIS) promoter is a frequent event in 3. Brash AR. Lipoxygenases: occurrence, functions, nase-2(15-LOX2)contributes to replicative senes- colorectal cancer and is associated with aneuploidy. catalysis, and acquisition of . J Biol Chem cense of human prostate progenitor cells. Oncogene Oncogene 2005;24:7320 ^ 6. 1999;274:23679 ^82. 2005;19:3583^95. 27. Lim H, Key SK. A novel pathway of prostacyclin 4. Peters-GoldenM,BailieM,MarshallT,etal.Protec- 16. Yang P, Cartwright C, Chan D,Vijjeswarapu M, Ding signaling-hanging out with nuclear receptors. tion from pulmonary fibrosis in leukotrienes-deficient J, Newman RA. Zyflamend (R)-mediated inhibition of Endocrinology 2002;143:3207 ^ 10. mice. Am J Respir Crit Care Med 2002;165:229 ^ 35. human prostate cancer PC3 cell proliferation: effects 28. Brown JR, DuBois RN. COX-2: a molecular target 5. Tilley SL, Coffman TM, Koller BH. Mixed messages: on 12-LOX and Rb protein phosphorylation. Cancer for colorectal cancer prevention. J Clin Oncol 2005; modulation of inflammation and immune responses Biol Ther 2007;6:228 ^36. 23:2840^55. by prostaglandins and . J Clin Invest 17. Yang P, Chan D, Felix E, et al. Determination of 29. Su B, Guan M, Zhao R, LuY. Expression of prosta- 2001;108:15^23. endogenous tissue inflammation profiles by LC/ glandin D synthase in ovarian cancer. Clin Chem Lab 6. Toda A,YokomizoT, ShimizuT. recep- MS/MS:COX- and LOX-derived bioactive lipids. Med 2001;39:1198 ^ 203. tors. Prostaglandins Other Lipid Mediat 2002;68 ^ 69: Prostaglandins Leukot Essent Fatty Acids 2006; 30. Nie D, Che M, Zacharek A, et al. Differential ex- 575 ^ 85. 75:385 ^ 95. pression of thromboxane synthase in prostate carci- 7. Tsuboi K, SugimotoY, Ichikawa A. Prostanoid recep- 18. Wang E, Miller L, Ohnmacht GA, Liu E, Marincola noma: role in cell motility. Am J Pathol 2004;164: tor subtypes. Prostaglandins Other Lipid Mediat FM. High fidelity mRNA amplification for gene profil- 429 ^ 39. 2002;68 ^ 69:535 ^ 56. ing. Nat Biotechnol 2000;18:457 ^ 9. 31.Watkins G, Douglas-Jones A, Mansel RE, Jiang WG. 8. Evans JH, Spencer DM, Zweifach A, Leslie CC. Intra- 19. Gupta RA, Tejada LV,Tong BJ, et al. Cyclooxyge- Expression of thromboxane synthase,TBXAS1and the cellular calcium signals regulating cytosolic phospho- nase-1 is overexpressed and promotes angiogenic thromboxane A2receptor, TBXA2R, in human breast lipase A2translocation to internal membranes. J Biol growth factor production in ovarian cancer. Cancer cancer. Int Semin Surg Oncol 2005;2:23. Chem 2001;276:30150 ^ 60. Res 2003;63:906 ^ 11. 32. Ermert L, Dierkes C, Ermert M. Immunohistochemi- 9. Wang E, Ngalame Y, Panelli MC, et al. Peritoneal and 20. Ross DT, Scherf U, Eisen MB, et al. Systematic cal expression of coclooxygenase isoenzymes and sub-peritoneal stroma may facilitate regional spread of variation in gene expression patterns in human cancer downstream enzymes in human lung tumors. ovarian cancer. Clin Cancer Res 2005;11:113^ 22. cell lines. Nat Genet 2000;24:227 ^ 35. Clin Cancer Res 2003;9:1604 ^10. 10. Wang X, Deavers M, Patenia R, et al. Monocyte/ 21. KinoY,KojimaF,KiguchiK,IgarashiR,IshizukaB, 33. Meng ZX, SunJX, LingJJ, et al. Prostaglandin E(2) macrophage and T-cell infiltrates in peritoneum of Kawai S. Prostaglandin E2production in ovarian regulates Foxo activity via Akt pathway: implications patients with ovarian cancer or benign pelvic disease. cancer cell lines is regulated by cyclooxygenase-1, for pancreatic islet h cell dysfunction. Diabetologia J Transl Med 2006;4:30. not cyclooxygenase-2. Prostaglandins Leukot Essent 2006;49:2959 ^ 68. 11. Gordon IO, Freedman RS. Defective antitumor function Fatty Acids 2005;73:103 ^ 11. 34. Yamaki T, Endoh K, Miyahara M, et al. Prosta- of monocyte-derived macrophages from epithelial ovar- 22. Myung S-J, Rerko RM, Yan M, et al. 15-Hydroxy- glandin E2activates Src signaling in lung adeno- ian cancer patients. Clin Cancer Res 2006;12:1515^ 24. prostaglandin dehydrogenase is an in vivo suppressor carcinoma cell via EP3. Cancer Lett 2004;214: 12. Kempen EC, Yang P, Felix E, Madden T, Newman of colon tumorigenesis. Proc Natl Acad Sci U S A 115 ^ 20 . RA. Simultaneous quantification of arachidonic ac- 2006;103:12098^102. 35. VignatiS,AlbertiniV,RinaldiA,etal.Cellularand id metabolites in cultured tumor cells using high- 23. Bhatia B, Maldonado CJ,Tang S, et al. Subcellular molecular consequences of peroxisome proliferators- performance liquid chromatography/electrospray localization and tumor-suppressive functions of activated receptor-gamma activation in ovarian cancer ionization tandem mass spectrometry. Anal Biochem 15-lipoxygenase 2(15-LOX2)and its splice variants. cells. Neoplasia 2006;8:851 ^ 61. 2001;297:183^90. J Biol Chem 2003;278:25091 ^ 100. 36. Yang P, Chan D, Felix E, et al. Formation and anti- 13 . YangP,FelixE,MaddenT,FischerSM,NewmanRA. 24. Furstenberger G, Krieg P,Muller-Decker K, Habenicht proliferative effect of prostaglandin E(3) from eicosa- Quantitative high-performance liquid chromatography/ AJR. What are cyclooxygenases and lipoxygenases pentaenoic acid in human lung cancer cells. J Lipid electrospray ionization tandem mass spectrometric anal- doing in the driver’s seat of carcinogenesis? IntJCancer Res 2004;45:1030^9. ysis of 2- and 3-series prostaglandins in cultured tumor 2006;119:2247 ^ 54. 37. Clark JD,TamS. Potential therapeutic uses of phos- cells. Anal Biochem 2001;308:168^90. 25. JiangJ-G,ChenC-L,CardJW,etal.Cytochrome pholipaseA2inhibitors. Expert OpinTher Pat 2004;14: 14. Shureiqi I,WuY, Chen D, et al. The critical role of 15- P450 2J2 promotes the neoplastic phenotype of 937 ^ 50.

Clin Cancer Res 2007;13(19) October 1, 2007 5744 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Comparative Analysis of Peritoneum and Tumor Eicosanoids and Pathways in Advanced Ovarian Cancer

Ralph S. Freedman, Ena Wang, Sonia Voiculescu, et al.

Clin Cancer Res 2007;13:5736-5744.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/13/19/5736

Cited articles This article cites 37 articles, 11 of which you can access for free at: http://clincancerres.aacrjournals.org/content/13/19/5736.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/13/19/5736.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/13/19/5736. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research.