Arachidonic Acid Pathway Members PLA2G7, HPGD, EPHX2, and CYP4F8 Identified as Putative Novel Therapeutic Targets in Prostate Cancer
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.002
ISSN1525-2191
AutoresPaula Vainio, Santosh Gupta, Kirsi Ketola, Tuomas Mirtti, John-Patrick Mpindi, Pekka Kohonen, Vidal Fey, Merja Perälä, Frank Smit, Gerald W. Verhaegh, Jack A. Schalken, Kalle Alanen, Olli Kallioniemi, Kristiina Iljin,
Tópico(s)Prostate Cancer Treatment and Research
ResumoThe arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress. The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress. Androgen deprivation has remained one of the main therapeutic options for prostate cancer; however, hormonal therapy is not curative, often resulting in the development of castration-resistant prostate cancer. Such recurrent and often metastatic tumors remain virtually impossible to treat with current medications.1Tannock I.F. de Wit R. Berry W.R. Horti J. Pluzanska A. Chi K.N. Oudard S. Théodore C. James N.D. Turesson I. Rosenthal M.A. Eisenberger M.A. TAX 327 InvestigatorsDocetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer.N Engl J Med. 2004; 351: 1502-1512Crossref PubMed Scopus (4671) Google Scholar Recent studies indicate that prostate tumors may adapt to the reduced levels of testosterone by acquiring hypersensitivity to low steroid levels [eg, by mutations or amplifications of the androgen receptor (AR)], as well as by increased intracrine synthesis of androgens.2Holzbeierlein J. Lal P. LaTulippe E. Smith A. Satagopan J. Zhang L. Ryan C. Smith S. Scher H. Scardino P. Reuter V. Gerald W.L. Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance.Am J Pathol. 2004; 164: 217-227Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 3Pienta K.J. Bradley D. Mechanisms underlying the development of androgen-independent prostate cancer.Clin Cancer Res. 2006; 12: 1665-1671Crossref PubMed Scopus (326) Google Scholar, 4Taichman R.S. Loberg R.D. Mehra R. Pienta K.J. The evolving biology and treatment of prostate cancer.J Clin Invest. 2007; 117: 2351-2361Crossref PubMed Scopus (107) Google Scholar, 5Dillard P.R. Lin M.F. Khan S.A. Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol.Mol Cell Endocrinol. 2008; 295: 115-120Crossref PubMed Scopus (169) Google Scholar Novel drugs targeting de novo intratumoral steroid synthesis are under development to increase the efficacy of hormonal treatments.6Attard G. Reid A.H. Olmos D. de Bono J.S. Antitumor activity with CYP17 blockade indicates that castration-resistant prostate cancer frequently remains hormone driven.Cancer Res. 2009; 69: 4937-4940Crossref PubMed Scopus (141) Google Scholar Nonetheless, in addition to these therapies, rationally designed novel therapeutic approaches are needed. The arachidonic acid (AA) pathway, a key inflammatory pathway involved in cellular signaling, is implicated in prostate carcinogenesis.7Patel M.I. Kurek C. Dong Q. The arachidonic acid pathway and its role in prostate cancer development and progression.J Urol. 2008; 179: 1668-1675Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Arachidonic acid is stored in cell membranes, but on stimulation it is mobilized by phospholipase A2 (PLA2) and is converted to various biologically active eicosanoids by cyclooxygenases (COXs), lipoxygenases (LOXs), or P450 cytochromes (CYP). The rate of AA turnover in prostate cancer cells is 10-fold enhanced, compared with the surrounding normal prostate epithelial cells,8Chaudry A. McClinton S. Moffat L.E. Wahle K.W. Essential fatty acid distribution in the plasma and tissue phospholipids of patients with benign and malignant prostatic disease.Br J Cancer. 1991; 64: 1157-1160Crossref PubMed Scopus (77) Google Scholar and AA, as well as many eicosanoids, induces prostate cancer proliferation in vitro.7Patel M.I. Kurek C. Dong Q. The arachidonic acid pathway and its role in prostate cancer development and progression.J Urol. 2008; 179: 1668-1675Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9Wang Y. Corr J.G. Thaler H.T. Tao Y. Fair W.R. Heston W.D. Decreased growth of established human prostate LNCaP tumors in nude mice fed a low-fat diet.J Natl Cancer Inst. 1995; 87: 1456-1462Crossref PubMed Scopus (219) Google Scholar, 10Ghosh J. Myers C.E. Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase.Biochem Biophys Res Commun. 1997; 235: 418-423Crossref PubMed Scopus (223) Google Scholar The specific molecular mechanisms involved in this process and the role of individual genes along the AA pathway remain poorly understood. Recently, AA synthesis has been shown to induce androgen production in steroid-starved prostate cancer cells, suggesting a contribution also to the activation of AR in castration-resistant prostate cancer progression.11Locke J.A. Guns E.S. Lehman M.L. Ettinger S. Zoubeidi A. Lubik A. Margiotti K. Fazli L. Adomat H. Wasan K.M. Gleave M.E. Nelson C.C. Arachidonic acid activation of intratumoral steroid synthesis during prostate cancer progression to castration resistance.Prostate. 2010; 70: 239-251PubMed Google Scholar Previously, prostate carcinogenesis and cancer growth have been linked mostly to aberrant AA metabolism through COX and LOX pathways.12Fürstenberger G. Krieg P. Müller-Decker K. Habenicht A.J. What are cyclooxygenases and lipoxygenases doing in the driver's seat of carcinogenesis?.Int J Cancer. 2006; 119: 2247-2254Crossref PubMed Scopus (130) Google Scholar, 13Matsuyama M. Yoshimura R. The target of arachidonic acid pathway is a new anticancer strategy for human prostate cancer.Biologics. 2008; 2: 725-732PubMed Google Scholar, 14Wang D. Dubois R.N. Eicosanoids and cancer.Nat Rev Cancer. 2010; 10: 181-193Crossref PubMed Scopus (1155) Google Scholar Widely used COX-2 inhibitors suppress the growth of prostate cancer cells in vitro and tumorigenesis in vivo.15Hsu A.L. Ching T.T. Wang D.S. Song X. Rangnekar V.M. Chen C.S. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2.J Biol Chem. 2000; 275: 11397-11403Crossref PubMed Scopus (634) Google Scholar, 16Narayanan B.A. Narayanan N.K. Pittman B. Reddy B.S. Regression of mouse prostatic intraepithelial neoplasia by nonsteroidal anti-inflammatory drugs in the transgenic adenocarcinoma mouse prostate model.Clin Cancer Res. 2004; 10: 7727-7737Crossref PubMed Scopus (99) Google Scholar, 17Patel M.I. Subbaramaiah K. Du B. Chang M. Yang P. Newman R.A. Cordon-Cardo C. Thaler H.T. Dannenberg A.J. Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism.Clin Cancer Res. 2005; 11: 1999-2007Crossref PubMed Scopus (181) Google Scholar However, because of cardiovascular adverse effects, the use of COX-2 inhibitors as cancer drugs raises safety concerns.18Kearney P.M. Baigent C. Godwin J. Halls H. Emberson J.R. Patrono C. Do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials.BMJ. 2006; 332: 1302-1308Crossref PubMed Scopus (1193) Google Scholar In addition, inhibitors of the ALOX5 (arachidonate 5-lipoxygenase, or 5-LO) and secretory phospholipase A2 (sPLA2) proteins have been shown to reduce prostate cancer cell proliferation in vitro,7Patel M.I. Kurek C. Dong Q. The arachidonic acid pathway and its role in prostate cancer development and progression.J Urol. 2008; 179: 1668-1675Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10Ghosh J. Myers C.E. Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase.Biochem Biophys Res Commun. 1997; 235: 418-423Crossref PubMed Scopus (223) Google Scholar, 19Moretti R.M. Montagnani Marelli M. Sala A. Motta M. Limonta P. Activation of the orphan nuclear receptor RORalpha counteracts the proliferative effect of fatty acids on prostate cancer cells: crucial role of 5-lipoxygenase.Int J Cancer. 2004; 112: 87-93Crossref PubMed Scopus (39) Google Scholar, 20Sved P. Scott K.F. McLeod D. King N.J. Singh J. Tsatralis T. Nikolov B. Boulas J. Nallan L. Gelb M.H. Sajinovic M. Graham G.G. Russell P.J. Dong Q. Oncogenic action of secreted phospholipase A2 in prostate cancer.Cancer Res. 2004; 64: 6934-6940Crossref PubMed Scopus (83) Google Scholar although these inhibitors have not yet progressed to clinical trials in prostate cancer. The AA pathway is a promising area for translational research, because many targets along this pathway have been already intensively investigated in other indications, such as cardiovascular diseases and pain, providing an opportunity for repositioning of drugs already in clinical development to new indications. Furthermore, understanding the roles of different downstream pathways and individual enzymes in AA metabolism may provide more effective therapeutic opportunities with fewer adverse effects.14Wang D. Dubois R.N. Eicosanoids and cancer.Nat Rev Cancer. 2010; 10: 181-193Crossref PubMed Scopus (1155) Google Scholar In the present study, we applied bioinformatics to systematically explore the expression patterns of 36 key AA pathway members in vivo, and then performed targeted clinical validation and functional siRNA knockdown studies of the seven most prostate cancer-specific AA pathway genes. The results point to four previously undescribed potential therapeutic targets in prostate cancer: PLA2G7, HPGD, EPHX2, and CYP4F8. A list of 36 central AA pathway members was collected from multiple studies on AA pathway in prostate cancer.7Patel M.I. Kurek C. Dong Q. The arachidonic acid pathway and its role in prostate cancer development and progression.J Urol. 2008; 179: 1668-1675Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Chaudry A.A. Wahle K.W. McClinton S. Moffat L.E. Arachidonic acid metabolism in benign and malignant prostatic tissue in vitro: effects of fatty acids and cyclooxygenase inhibitors.Int J Cancer. 1994; 57: 176-180Crossref PubMed Scopus (136) Google Scholar, 22Endsley M.P. Thill R. Choudhry I. Williams C.L. Kajdacsy-Balla A. Campbell W.B. Nithipatikom K. Expression and function of fatty acid amide hydrolase in prostate cancer.Int J Cancer. 2008; 123: 1318-1326Crossref PubMed Scopus (73) Google Scholar, 23Jain S. Chakraborty G. Raja R. Kale S. Kundu G.C. Prostaglandin E2 regulates tumor angiogenesis in prostate cancer.Cancer Res. 2008; 68: 7750-7759Crossref PubMed Scopus (133) Google Scholar The GeneSapiens database24Kilpinen S. Autio R. Ojala K. Iljin K. Bucher E. Sara H. Pisto T. Saarela M. Skotheim R.I. Björkman M. Mpindi J.P. Haapa-Paananen S. Vainio P. Edgren H. Wolf M. Astola J. Nees M. Hautaniemi S. Kallioniemi O. Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues.Genome Biol. 2008; 9: R139Crossref PubMed Scopus (214) Google Scholar was then applied to bioinformatically explore the gene expression levels across 9783 human tissue samples. Briefly, GeneSapiens (http://www.genesapiens.org/) is a collection of 9873 Affymetrix microarray experiments. All data are reannotated and normalized with a custom algorithm. The data are collected from various publicly available sources, including Gene Expression Omnibus and ArrayExpress. The data cover 175 different tissue types.24Kilpinen S. Autio R. Ojala K. Iljin K. Bucher E. Sara H. Pisto T. Saarela M. Skotheim R.I. Björkman M. Mpindi J.P. Haapa-Paananen S. Vainio P. Edgren H. Wolf M. Astola J. Nees M. Hautaniemi S. Kallioniemi O. Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues.Genome Biol. 2008; 9: R139Crossref PubMed Scopus (214) Google Scholar Mean expression of each AA pathway gene was determined in prostate cancer (n = 329), healthy prostate (n = 147), and all normal tissue samples (n = 1626). Genes with (1) significantly higher expression in prostate cancer compared with normal prostate (fold change FC > 2, P < 0.001) and (2) genes showing high expression (FC > 1.5 and P << 0.001 or FC > 2 and P < 0.001) in prostate cancer compared with mean expression across all healthy tissues were selected for further studies. Primary prostate cancer samples derived from total prostatectomy patients (n = 33, see Supplemental Table S1 at http://ajp.amjpathol.org), nonmalignant samples with normal histology (n = 17) and hyperplastic histology (benign prostatic hyperplasia) (n = 5) and tissue microarrays containing metastatic prostate cancer samples (n = 103) from 62 patients were obtained from the Department of Pathology at Turku University Hospital. The nonmalignant samples used for immunohistochemistry were from patients aged 49–86 years (mean, 70.6 years). The 19 advanced prostate tumor samples used in quantitative reverse transcription PCR (qRT-PCR) have been described previously.25Iljin K. Wolf M. Edgren H. Gupta S. Kilpinen S. Skotheim R.I. Peltola M. Smit F. Verhaegh G. Schalken J. Nees M. Kallioniemi O. TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming.Cancer Res. 2006; 66: 10242-10246Crossref PubMed Scopus (186) Google Scholar All tissue samples were used according to contemporary regulatory guidelines. Gene expression in clinical samples and siRNA-induced target gene silencing were validated with qRT-PCR. For the primary prostate cancer tissue samples (n = 33) obtained from the Department of Pathology at Turku University Hospital, frozen tissue blocks were sectioned and hematoxylin and eosin staining was used for confirmation and localization of cancerous tissue. Skin biopsy equipment was then used to collect cancer samples for RNA extractions. The histology of the three normal prostate tissue samples was also confirmed to be free of any pathological alteration. RNA samples extracted with an RNeasy mini kit (Qiagen, Valencia, CA) were reverse-transcribed to cDNA (high capacity cDNA reverse transcription kit; Applied Biosystems, Foster City, CA) and TaqMan qRT-PCR was performed with an Applied Biosystems 7900HT instrument (Finnish DNA Microarray Centre, Turku Centre For Biotechnology, University of Turku, Finland). The primers and probes used are listed in Table 1. The results were analyzed with the manufacturer's software packages (sequence detection system SDS 2.3 with RQ relative quantification software; Applied Biosystems). β-Actin was used as an endogenous control. In clinical samples, relative mRNA expression for each gene in the normal control tissue samples (n = 3) was set as 1 (mean relative expression).Table 1TaqMan qRT-PCR Primers and Probes Designed Using Roche Universal ProbeLibrary Assay Design Center and Used to Validate Target Gene Silencing by siRNAs and mRNA Expression in Clinical Prostate Cancer SamplesGeneForward primerReverse primerProbeALOX15B5′-TGAGGTCTTCACCCTGGCTA-3′5′-TTGATGTGCAGGGTGTATCG-3′43AR5′-GCCTTGCTCTCTAGCCTCAA-3′5′-GTCGTCCACGTGTAAGTTGC-3′14CYP4F85′-CATCTTCAGCTTTGACAGCAA-3′5′-TGAGCTCCATGATCGCAGTA-3′2EPHX25′-TTCTGCTGGACACCCTGAA-3′5′-TTCAGATTAGCCCCGATGTC-3′45ERG5′-CAGGTGAATGGCTCAAGGA-3′5′-AGTTCATCCCAACGGTGTCT-3′44FAAH5′-CTCTGCTGCCAAGGCTGT-3′5′-TGCAGTTCCCAGAGTTTTCC-3′73HPGD5′-TGGTCAATAATGCTGGAGTGA-3′5′-GGTTCCACTGATAACAGAAACCA-3′48KIF115′-CATCCAGGTGGTGGTGAGAT-3′5′-TATTGAATGGGCGCTAGCTT-3′53MT1X5′-CTTCTCCTTGCCTCGAAATG-3′5′-ACAGGCACAGGAGCCAAC-3′15MT2A5′-CTAGCCGCCTCTTCAGCA-3′5′-GCAGGTGCAGGAGTCACC-3′68PLA2G2A5′-ACCTGCCCTGTCTCCAAAC-3′5′-TTTGTTCTGCACTCCTGCTC-3′32PLA2G75′-TGGCTCTACCTTAGAACCCTGA-3′5′-TTTTGCTCTTTGCCGTACCT-3′63PLK15′-CACAGTGTCAATGCCTCCA-3′5′-TTGCTGACCCAGAAGATGG-3′30 Open table in a new tab The results are presented as means ± SD. Statistical analyses were performed using Student's t-test (P < 0.05, P < 0.01, and P < 0.001) and Pearson's correlation coefficient, unless otherwise indicated. Acetone-fixed frozen sections (6 μm) of primary prostate cancer samples were dried and endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide (H2O2). Goat serum was used to prevent unspecific staining. The slides were incubated with primary antibodies affinity-purified IgG to human PLA2G7 protein (1:200; Cayman Chemical, Ann Arbor, MI) or to HPGD (1:400; PA005679; Sigma-Aldrich, St. Louis, MO) at 4°C overnight. After Tris-buffered saline washes, the slides were incubated with biotinylated goat anti-rabbit secondary antibody (1:200; Vectastain; Vector Laboratories, Burlingame, CA), followed by Vectastain ABC reagent incubation and diaminobenzidine staining. Mayer's hematoxylin was used in counterstaining. The paraffin-mounted metastatic prostate cancer tissue microarrays and the paraffin-mounted tissue samples containing histologically normal (n = 14) and hyperplastic (n = 5) prostate were stained using a Lab Vision autostainer (Thermo Fisher Scientific, Fremont, CA) and PowerVision+ Poly-HRP immunohistochemistry detection system kit reagents (ImmunoVision Technologies, Burlingame, CA). Samples were deparaffinized and antigen retrieval was performed in Tris-buffered saline (Dako Target retrieval solution, pH 9; Dako, Glostrup, Denmark) using microwaving for 2 × 7 minutes. Endogenous peroxidase activity was blocked with 3% H2O2, and slides were incubated with dilution buffer (Dako) for 10 minutes before incubation with the primary antibodies (1:200–1:400) for 1 hour. After washing and 20 minutes of PowerVision post-blocking, slides were incubated with PowerVision Poly-HRP anti-rabbit IgG for 30 minutes and with diaminobenzidine for 10 minutes. Mayer's hematoxylin was used in counterstaining, and slides were coated with coverslips after ethanol series and xylene. Protein expression in each cancer sample was graded into four groups, based on staining intensity (−, +, ++, and +++). An Olympus BX50 microscope (Olympus, Tokyo, Japan), Nikon ACT-1 software version 2.62 (Nikon, Tokyo, Japan), and a digital camera (DXM1200; A.G. Heinze, Lake Forest, CA) were used in the photographing. LNCaP androgen-sensitive human prostate adenocarcinoma cells26Harris S.E. Rong Z. Harris M.A. Lubahn D.B. Androgen receptor in human prostate carcinoma LNCaP/ADEP cells contains a mutation which alters the specificity of the steroid dependent transcriptional activation region.Program and Abstracts, 72nd Annual Meeting of the Endocrine Society, Atlanta, GA. 1990; : 93Google Scholar were provided by Dr. Marco Cecchini (University of Bern, Switzerland) and were grown in T-Medium (Invitrogen, Carlsbad, CA). VCaP vertebral prostate cancer cells27Korenchuk S. Lehr J.E. MClean L. Lee Y.G. Whitney S. Vessella R. Lin D.L. Pienta K.J. VCaP, a cell-based model system of human prostate cancer.In Vivo. 2001; 15: 163-168PubMed Google Scholar were received from Drs. Adrie van Bokhoven (University Medical Center, Nijmegen, Netherlands) and Kenneth Pienta (University of Michigan, MI) and were maintained in RPMI-1640. For the RNAi studies, siRNAs (HP GenomeWide; Qiagen) were plated onto 384-well plate (Greiner Bio-One, Frickenhausen, Germany), followed by addition of the transfection agent (siLentFect lipid reagent; Bio-Rad Laboratories, Hercules, CA) in Opti-MEM medium (Invitrogen) and an appropriate quantity of cells (1500–2000 per well). The final siRNA concentration was 13 nmol/L. After 48 hours (VCaP) or 72 hours (LNCaP) incubation, cells were assayed with CellTiter-Blue (Promega, Madison WI) for viability and ApoONE (Promega) for apoptosis according to the manufacturer's instructions. The EnVision multilabel plate reader (Perkin-Elmer, Waltham, MA) was used for signal quantification. The raw results were normalized using B-score,28Brideau C. Gunter B. Pikounis B. Liaw A. Improved statistical methods for hit selection in high-throughput screening.J Biomol Screen. 2003; 6: 634-647Crossref Scopus (244) Google Scholar and siRNAs reducing cell viability by ≥2 SD from the median of the controls (corresponding to P < 0.05) were considered putative antiproliferative siRNAs. AllStars negative control (scrambled siRNA; Qiagen) and lipid only were used as negative controls; siRNAs against KIF11 (kinesin family member 11) and PLK1 (polo-like kinase 1) were used as positive controls. VCaP cells were transfected with siRNAs as described above. At 48 hours after transfection, H2O2 (0, 100, 200, and 400 μmol/L) was added to the cells for 6 hours. Cells were washed and incubated for an additional 24 hours in normal media. Cell viability was determined with CellTiter-Glo (Promega) according to the manufacturer's instructions. Western blot analysis was performed to validate the antibodies used in immunohistochemical analysis (see Supplemental Figure S1 at http://ajp.amjpathol.org) and to visualize the possible changes in AR and prostate-specific antigen (PSA) expression in response to target inhibition. Cells were lysed and proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Antibodies against AR (1:1000, NeoMarkers, Fremont, CA), PSA (1:1000, Dako), platelet activating factor (PAF) acetylhydrolase (encoded by PLA2G7) (1:500, Cayman Chemical), 15-PGDH (encoded by HPGD) (1:500, Sigma-Aldrich), and β-actin (1:5000, mouse-monoclonal, Becton Dickinson, Franklin Lakes, NJ) were used. Signal was detected with 1:4000 dilution of horseradish peroxidase-conjugated secondary antibodies (Invitrogen Molecular Probes, Carlsbad, CA), followed by visualization with an enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). Anti-androgen flutamide was purchased from Sigma-Aldrich and diluted in ethanol. LNCaP cells were plated and transfected with siRNAs as described above 24 hours before addition of 10 μmol/L flutamide or ethanol control. Cell viability was determined with CellTiter-Glo (Promega) after 48 hours treatment. We first performed a detailed bioinformatic analysis of the mRNA expression levels for all 36 central AA pathway genes in both prostate cancer and healthy prostate, as well as in all normal tissues present in the GeneSapiens database (Figure 1A and Supplemental Table S2 at http://ajp.amjpathol.org). Six genes (ALOX15B, CYP4F8, EPHX2, FAAH, PLA2G2A, and PLA2G7) were highly expressed in prostate cancer samples, compared with expression levels in the normal tissues studied. ALOX15B, CYP4F8, EPHX2, FAAH, and PLA2G2A showed more prostate-specific than prostate cancer-specific expression, whereas PLA2G7 mRNA levels were clearly elevated in prostate cancer, compared with normal prostate. In addition, HPGD mRNA expression was significantly elevated in a subset of prostate cancer samples, compared with normal prostate, even though the mean expression level was not significantly different in prostate cancer in comparison to all normal tissue types. These seven genes were selected for further studies. The expression profiles for these genes across all 43 healthy and 68 malignant tissue types, as well as in prostate cancer samples compared with normal prostate samples, are presented in Supplemental Figure S2 (available at http://ajp.amjpathol.org). Interestingly, high HPGD expression was associated with metastatic prostate cancer, a finding supported also by independent datasets in Oncomine29Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. ONCOMINE: a cancer microarray database and integrated data-mining platform.Neoplasia. 2004; 6: 1-6Abstract Full Text PDF PubMed Google Scholar (see Supplemental Figure S3 at http://ajp.amjpathol.org); no such enrichment in advanced disease was found for the other six genes (data not shown). An overview of the roles of the seven genes in the AA cascade is presented in Figure 1B. Three of the genes (PLA2G2A, FAAH, and ALOX15B) have previously been reported to regulate prostate cancer growth. The PLA2G2A protein (group IIA phospholipase A2, a member of the secretory phospholipase A2 family) is a gatekeeper for the pathway releasing AA from the membrane phospholipids. The FAAH protein, fatty-acid amide hydrolase 1, increases AA concentrations by hydrolyzing endocannabinoids. Inhibition of either one of these enzymes is known to decrease prostate cancer cell growth.20Sved P. Scott K.F. McLeod D. King N.J. Singh J. Tsatralis T. Nikolov B. Boulas J. Nallan L. Gelb M.H. Sajinovic M. Graham G.G. Russell P.J. Dong Q. Oncogenic action of secreted phospholipase A2 in prostate cancer.Cancer Res. 2004; 64: 6934-6940Crossref PubMed Scopus (83) Google Scholar, 22Endsley M.P. Thill R. Choudhry I. Williams C.L. Kajdacsy-Balla A. Campbell W.B. Nithipatikom K. Expression and function of fatty acid amide hydrolase in prostate cancer.Int J Cancer. 2008; 123: 1318-1326Crossref PubMed Scopus (73) Google Scholar, 31Dong Q. Patel M. Scott K.F. Graham G.G. Russell P.J. Sved P. Oncogenic action of phospholipase A2 in prostate cancer.Cancer Lett. 2006; 240: 9-16Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar In contrast, The ALOX15B protein (arachidonate 15-lipoxygenase B) metabolizes AA to 15-hydroxyeicosatetraenoic acid (HETE) and has been described as a functional tumor suppressor in prostate cancer.32Tang D.G. Bhatia B. Tang S. Schneider-Broussard R. 15-Lipoxygenase 2 (15-LOX2) is a functional tumor suppressor that regulates human prostate epithelial cell differentiation, senescence, and growth (size).Prostaglandins Other Lipid Mediat. 2007; 82: 135-146Crossref PubMed Scopus (42) Google Scholar Four other genes (ie, PLA2G7, CYP4F8, EPHX2, and HPGD) have not previously been shown to be functionally involved in prostate cancer and were therefore of special interest. The PLA2G7 protein (platelet-activating factor acetylhydrolase, or PAF-acetylhydrolase; also known as LDL-associated phospholipase 2), is a potent pro- and antiinflammatory enzyme with the ability to degrade PAF and truncated membrane phospholipids generated by oxidative stress.33Stafforini D.M. Biology of platelet-activating factor acetylhydrolase (PAF-AH, lipoprotein associated phospholipase A2).Cardiovasc Drugs Ther. 2009; 23: 73-83Crossref PubMed Scopus (164) Google Scholar Based on enzymatic assays, the CYP4F8 protein (cytochrome P450 4F8) has been proposed to oxygenate and hydroxylate COX-derived products to 19-hydroxy-PGE2 (prostaglandin E2).34Bylund J. Hidestrand M. Ingelman-Sundberg M. Oliw E.H. Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of prostaglandin endoperoxides.J Biol Chem. 2000; 275: 21844-21849Crossref PubMed Scopus (75) Google Scholar The EPHX2 protein (epoxide hydrolase 2) degrades AA-derived and CYP-produced bioactive epoxy fatty acids and has been suggested as a potential metastasis suppressor gene in breast cancer.35Thomassen M. Tan Q. Kruse T.A. Gene expression meta-analysis identifies chromosomal regions and candidate genes involved in breast cancer metastasis [Erratum appeared in Breast Cancer Res Treat 2009, 113:251–252].Cancer Res Treat. 2009; 113: 239-249Crossref Scopus (83) Google Scholar On the other hand, the HPGD protein [15-hydroxyprostaglandin dehydrogenase (NAD+), or 15-PGDH] inactivates eicosanoids, mainly prostaglandins, and it has also been suggested to be a tumor suppressor.36Wolf I. O'Kelly J. Rubinek T. Tong M. Nguyen A. Lin B.T. Tai H.H. Karlan B.Y. Koeffler
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