Inactivation of tumor suppressors LKB1 and PTEN by arachidonic acid metabolites

This dissertation describes the modification and inactivation of tumor suppressors by arachidonic acid (AA) metabolites. Cyclooxygenase (COX) and lipoxygenase (LOX) enzymes are AA metabolizing enzymes involved in inflammation as well as cancer. In this work, a novel mechanism linking inflammation and cancer is presented. LKB1 is a serine/threonine kinase that activates AMP-Kinase, thereby regulating anabolic and catabolic processes in the cell. LKB1 has a nucleophilic cysteine in its activation loop. Chapter 2 demonstrates the covalent modification of LKB1 by clectrophilic, cyclopcntcnone prostaglandins. Formation of this covalent adduct in the activation loop inhibits the kinase activity of LKB1 and prevents the phosphorylation and activation of its substrate AMPK. This affects downstream signaling to important enzymes such as Aeetyl-CoA Carboxylase and S6-Kinase, preventing an appropriate response to low energy levels. PTEN is a phosphoinositol phosphatase and tumor suppressor involved in regulating the PI3K-Akt pathway. Chapter 3 illustrates the oxidation of PTEN by reactive oxygen lipid species generated during AA metabolism by COX-2 and 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased PIP3 production, activation of Akt, and phosphorylation of downstream Akt targets including GSK-3[3 and S6K. These effects are reeapitulated with pancreatic PLA2, which hydrolyzes the release of membrane-bound AA. Chapter 4 describes the covalent modification and inactivation of PTEN by cyclopentenone prostaglandins. Chapter 3 and 4 illustrate that covalent modification and oxidation of tumor suppressors during AA metabolism are not exclusive, but may both occur and contribute tumor suppressor inactivation. In summary, this work investigates two novel mechanisms involving the chemical inactivation of tumor suppressors at the protein level. Interference with the physiological roles of tumor suppressors may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids. These events likely occur as the results of constant exposure to eicosanoid metabolites and byproducts during chronic inflammation or during COX/LOX overexpression, giving rise to decreased sensitivity to anti-growth signals and the promotion of tumorigenesis.

INACTIV A TION OF TUMOR SUPPRESSORS LKB 1 AND PTEN BY ARACHIDONIC ACID METABOLITES by Tracy Marie Covey A dissertation subn11tted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medicinal Chemistry The University of Utah August 2007 Copyright © Tracy Marie Covey 2007 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Tracy Marie Covey This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Date Chair: Frank A. Fitzpatri DaVId A. J es Darrell R. Davis C. Dale Poulter THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING .APPROV AL To the Graduate Council of the University of Utah: I have read the dissertation of Tracy Marie Covey in its final fonn and have found that (1) its fonnat, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Date Frank A. Fitzpatric Chair: Supervisory Committee Approved for the Major Defartnlent (112M .::;::>.. '. Chris M. Ireland Chair Approved for the Graduate Council ABSTRACT This dissertation describes the tnodification and inactivation of tumor suppressors by arachidonic acid (AA) metabolites. Cyc100xygenase (COX) and lipoxygenase (LOX) enzymes arc AA metabolizing enzymes involved in inflalnmation as well as cancer. In this work, a novel mechanisnl linking inflammation and cancer is presented. LKB I is a serine/threonine kinase that activates AMP-Kinase, thereby regulating anabolic and catabolic processes in the cell. LKB 1 has a nucleophilic cysteine in its activation loop. Chapter 2 demonstrates the covalent modification of LKB I by eleetrophilic, cyclopentenone prostaglandins. Fonnation of this covalent adduct in the activation loop inhibits the kinase activity of LKB I and prevents the phosphorylation and activation of its substrate AMPK. This affects downstream signaling to important enzymes such as Acetyl-CoA Carboxylase and S6-Kinase, preventing an appropriate response to low energy levels. PTEN is a phosphoinositol phosphatase and tumor suppressor involved in regulating the PI3K-Akt pathway. Chapter 3 illustrates the oxidation of PTEN by reactive oxygen lipid species generated during AA metabolism by COX-2 and 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased P1P3 production, activation of Akt, and phosphorylation of downstream Akt targets including GSK-3p and S6K. These effects arc recapitulated with pancreatic PLA2, which hydrolyzes the release of membrane-bound AA. Chapter 4 describes the covalent modification and inactivation of PTEN by cyclopentenone prostaglandins. Chapter 3 and 4 illustrate that covalent modification and oxidation of tumor suppressors during AA metabolism are not exclusive, but may both occur and contribute tumor suppressor inactivation. In summary, this work investigates two novel mechanisms involving the chemical inactivation of tumor suppressors at the protein level. Interference with the physiological roles of tumor suppressors may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids. These events likely occur as the results of constant exposure to eicosanoid metabolites and byproducts during chronic inflammation or during COX/LOX overexprcssioll, gIVIng rise to decreased sensitivity to anti-growth signals and the promotion of tunl0rigcnesis. V To my parents, Tom and Dar1ene Wagner, who have always believed in me, for their unending love and support. TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iv LIST OF FIGURES ............................................................................................................. x LIscr OF ABBREVIATIONS ........................................................................................... xii ACKNOWLEDGMENTS .............................................................................................. xvii Chapter INFLAMMATION AND CANCER ........................................................... 1 1.1 Introdllction ................................................................................ I 1.1.1 Inflamnlatory Response ............................................. 2 1.1.2 Inflammation and CanceL .......................................... 3 1.1.3 Eicosanoids ................................................................. 6 1.2 Cyclooxygenase EnzYITIeS ......................................................... 7 1 1 Cyclooxygenase Structure .......................................... 8 1 COX Function and Catalysis ..................................... 9 1.2.3 COX Regulation ...................................................... 10 1.2.4 COX Metabolites ..................................................... 1 1 1 COX and Cancer ...................................................... 15 1.3 Lipoxygcnase EnzYlTIeS ........................................................... 19 1 1 Lipoxygenase Structure ........................................... 22 1.3.2 Lipoxygenase Function and Catalysis ..................... 22 1.3.3 Lipoxygenase Regulation ......................................... 23 1.3.4 Lipoxygenase Metabolites ....................................... 24 l.3.5 Lipoxygenase and Cancer ........................................ 25 1.4 Tumor Suppressors .................................................................. 28 1.5 References ................................................................................ 34 2 REACTIVE LIPID SPECIES FROM CYCLOOXYGENASE-2 INACTIVATE LKB I/STK11 TUMOR SUPPRESSOR .......................... 46 3 REACTIVE OXYGEN SPECIES FROM CYCLOOXYGENASE-2 AND 5-LIPOXYGENASE INACTIVATE PTEN TUMOR SUPPRESSOR .... 54 3.1 Supplementary Information ..................................................... 64 3.2 References ................................................................................ 65 4 COVALENT MODIFICATION OF PTEN TUMOR SUPPRESSOR BY PROSTAGLANDIN J2: EVIDENCE FOR MECHANISTIC OVERLAP ................................................................................................. 66 4.1 Introduction .............................................................................. 66 4.2 Results ...................................................................................... 67 4.2.1 Modification of Proteins by Cyclopentenone Prostaglandins ........................................................ 67 4.2.2 PTEN is Modified by L112-PGh but Not PGA2 ••..•......••.••....••••••...••....•..•••.....•.....•.....•••..•••..•• 69 4.2.3 L112-PGh Increases Phosphorylation of (T308)Akt ......................................................... 71 4.2.4 J-Series Prostaglandins Potently Activate Akt. ......................................................................... 71 4.2.5 Dose Response of L112-PGh ... ............................. 76 4.2.6 Tinle Course of L112-PGJ2 .................................................... 76 4.2.7 Phosphorylation of Akt by L112-PGJ2 is Independent ofPPAR ........................................... 76 4.2.8 Activation of Akt by L112-PG.h is Dependent on PI3K .................................................................. 81 4.2.9 L112-PGh Increases Phosphorylation of Akt Substrates ............................................................... 81 4.2.10 L112-PGh Alters the Conformation of PTEN ......... 85 4.2.11 Rosiglitazone Increases PTEN Expression and Decreases the Effects of L112-PGh on Akt Phosphorylation ..................................................... 87 4.3 Discussion ................................................................................ 87 4.4 Materials and Methods ............................................................. 94 4.4.1 Materials ................................................................... 94 4.4.2 Cell Culture ............................................................... 94 4.4.3 Isolation of Proteins Covalently Labeled by PG-biotin ........................................................... 94 4.4.4 Identification of Reduced and Oxidized Forms ofPTEN ................................................................ 95 4.4.5 Effect of PGs on Akt Phosphorylation and Signaling .............................................................. 95 4.4.6 Statistics .................................................................... 96 4.5 References ................................................................................ 96 VIll 5 MODEL OF EICOSANOIDS IN NEOPLASIA: INACTIVATION OF TUMOR SUPPRESSORS AS A NOVEL MECHANISM ....................... 99 5.1 Introduction .............................................................................. 99 5.1.1 Model of Eicosanoids in Cancer: PGE2 .......•.••••.••• 101 5.1.3 Model ofEicosanoids in Cancer: Beyond PGE2 .••• 104 5.2 Model of Eicosanoids in Cancer: A Complex Event ............. 1 06 5.3 Chemical Inactivation of Tumor Suppressors ....................... .107 5.3.1 DirectAlkylation .................................................... 110 5.3.2 Indirect Alkylation .................................................. 1 10 5.3.3 Direct Oxidation ...................................................... I 13 5.4 Conclusions ............................................................................ 113 5.5 References .............................................................................. 114 IX LIST OF FIGURES Figure page 1.1 Chronic Inflammation Predisposes to Cancer.. ........................................... .4 1.2 Cyclooxygenase Pathway .......................................................................... 13 1.3 Peroxyl Radical and Aldehyde Fonnation During Peroxidation of Arachidonic Acid ....................................................................................... 17 1.4 Michael Addition ....................................................................................... 20 1.5 The 5-Lipoxygenase Pathway .................................................................... 26 1.6 Oxidation of Cysteine Residues ................................................................. 29 4.1 Covalent Modification of Proteins by cyPG-biotin ................................... 68 4.2 PTEN is Covalently Modified by ll12-PGh but not PGA] ...................... 70 4.3 ll12-PGh is a Potent Stimulator of Phospho-(T308)Akt. ........................ 72 4.4 The J-Series Prostaglandins ....................................................................... 73 4.5 The J-series of Prostaglandins are Potent Stimulators of Phosphorylation at (T308)Akt .................................................................. 75 4.6 Dose Response of ll12-PGh Affecting Phosphorylation of (T308)Akt .................................................................................................. 77 4.7 Tilne Course of ll12-PGh Affecting Phosphorylation of (T308)Akt.. ...... 79 4.8 The Increase in Phosphorylation of Akt by ll12-PGh is Independent of PP AR ................................................................................ 80 4.9 Phosphorylation of Akt by ll12-PGh is Sensitive to PI3K Inhibitors ....... 82 4.10 ll12-PGh Increased Signaling to Downstrearn Akt Substrates ................. 83 4.11 L\12-PGh Treatment Alters the Confonnation ofPTEN and Results in PTEN Degradation ............................................................. 86 4.12 Rosiglitazone Increases PTEN Expression and Decreases the Effect of L\ 12-PGh on Phosphorylation of Akt ................................... 88 4.13 Retinoblastoma Tumor Suppressor is Not Covalently Modified by CyPGs .................................................................................... 92 4.14 Covalent Modification of Kinases Depends on a Nucleophilic, Accessible Cysteine ............................................................. 93 5.1 Model of PGE2 in Cancer ........................................................................ 102 5.2 Mechanisms of Inactivating Tumor Suppressors by Eicosanoid Biosynthesis .......................................................................... 111 Xl L1 11M llmol 4-HNE 4-0NE AA ACe AICAR AMP AMPK ANOYA ATeC ATM ATP BME Bel-2 BSA e - terminus e LIST OF ABBREVIATIONS delta micromoJar micromole 4-hydroxy-nonenal 4-oxo-nonenal arachidonic acid acetyl coA carboxylase 5-al11inoimidazolc-4-carboxamide-l-~-D-ribofuranoside adenosine monophosphate adenosine monophosphate kinase analysis of variance American Type Culture Collection ataxia telangiectasia tnutated kinase adenosine triphosphate ~-l11ercapto-cthano I B cell lymphoma 2 bovine serum albumin carboxy terminus cysteine cAMP CO2 COX CpG cyPG cysLT DMSO DNA EGFR ERK FAP FBS FCS FLAP GAPDH GPx GSK3~ h H I leI HETE cyclic adenosine monophosphate carbon dioxide cyclooxygenase cytosine phosphodiester guanine cyclopentenone prostaglandin cysteinylleukotriene dimethyl sulfoxide deoxyribonucleic acid endothelial growth factor receptor extracellular signal-regulated kinase familial adenOlnatous polyposis coli fetal bovine serum fetal calf serum five lipoxygenase activating protein glyceraldehyde 3-phosphatase dehydrogenase glutathione peroxidase glycogen synthase-3~ kinase hour histidine hydrogen peroxide hydrochloric acid hydroxyecosatetraenoic acid Xlll HpETE HRP IKK IP K kDa LOX LT MAPK mm mM mTOR NA NF-KB NSAIDs siRNA STK P PAGE PBS PDK PG hydroperoxyecosatetraenoic acid horseradish peroxidase I kappa B kinase immunoprecipitation lysine kiloDalton lipoxygenase leukotriene mitogen activated protein kinase minute millimolar mammalian target of rapamycin neutravidin nuclear factor kappa B non-steroidal anti-inflammatory drugs small interfering ribonucleic acid serine threonine kinase phosphorylated polyacrylamide gel electrophoresis phosphate-buffered sal ine phosphoinositol 3-dependent kinase prostaglandin XIV PI3K PIP PJS pKa PLA PPAR PTEN PTP PVOF R Rb ROLS ROS s S S6K SOS sem T TNFa phosphoinositol 3-kinase phosphatidylinositol phosphate Peutz-Jeughers syndrome acid dissociation constant phospholipase peroxisome proliferator-activated receptors phosphatase and tens in homolog deleted on chromosome 10 protein tyrosine phosphatase polyvinylidene fluoride arglmne retinoblastoma reactive oxygen lipid species reactive oxygen species second senne S6 kinase sodiulTI dodecyl sulfate standard error of mean threonine tumor necrosis factor alpha xv TSC Tx VEGFR WT X Y tuberous sclerosis complex thromboxane vascular endothelial growth factor receptor wild type any an1ino acid tyrosine XVI ACKNOWLEDGMENTS First and foremost, I would like to thank my mentor, Frank Fitzpatrick. I have tremendous respect for Frank as both a scientist and a person. It was a great pleasure to complete my dissertation research in Frank's lab with his mentorship and guidance. I would also like to thank my committee members for their time and suggestions on my projects. I am grateful to the Fitzpatrick lab, both past and present members, for their support, encouragement, and help along the way. In particular, I would like to thank the co-authors on my papers for their help and expertise: James E. Mullalley and Kornelia Edes. I would like to express gratitude to the American Foundation for Pharmaceutical Education and the Multidisciplinary Cancer Research Training Program for pre-doctoral fellowships and the unique opportunities they have provided. I would like to thank the American Society for Biochemistry and the Nature Publishing Group for permission to re-print my articles from the Journal qj' Biological Chemistry and Oncogene, respectively. Finally, J would like to thank my family and friends for their support and love. I am particularly grateful to my husband, Regis, who has always had the right mix of encouragement, patience, and humor during my graduate work and thesis writing. CHAPTER 1 INFLAMMATION AND CANCER 1.1 Introduction There is substantial evidence supporting the conclusion that chronic inflammation can predispose one to cancer. It is estimated that over 150/0 of cancers worldwide arise from chronic inflammation caused by an infection; this translates to a tumorigenic burden of nearly 1.2 million cases per year globally (1). Inflammation can be caused by a variety of factors including bacterial, viral, parasitic infections, nondigestible particles, chemical irritants, or environnlental toxins. This inflatnmation can become chronic as a result of either persistent inflammatory stimuli or aberrant mechanisms involved in resolving that inflammation. Epidelniologic and clinical research indicate that the longer the inflatnmation exists, the greater the risk of carcinogenesis. Chronic inflammation is implicated in the development of numerous types of cancers. For exanlple, autoimmune inflammatory bowel diseases, ulcerative colitis, and Crohn' s disease predispose to the development of cancers in the large intestine and terminal ileum (2-5); chronic pancreatitis leads to an increased risk of developing pancreatic cancer (6, 7); asbestos exposure leads to inflammation in the lung and increases lung cancer and mesothelioma incidence (8); chronic infection with the bacterium Hc1icobacter pylori can cause gastritis predisposing to gastric adenocarcinoma and an unusual form of gastric lymphoma (9, 10); parasitic infection with schistosonles and other trematodes causes cancers of the 2 urmary bladder and the hepatic biliary tract (11, 12); hunlan papillomavirus (HPV) infections cause nearly 900/0 of all the cases of cervical cancer (13, 14); persistent hepatitis infections cause inflammation of liver tissue and an increased risk of liver cancer (15). It is clear that chronic inflammation provides a lTIilieu conducive to the development of cancer and the causal relationship between inflammation and cancer is widely accepted. However, the exact molecular and cellular mechanisms of inflammation pronl0ting the initiation and progression of cancer remain uncertain. This dissertation work investigates the inactivation of tumor suppressors by arachidonic acid (AA) metabolislTI as a potential mechanism linking inflammation and cancer. The main point of this chapter is to describe cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, their metabolites, and the roles they play in inflammation and cancer. An introduction to tumor suppressor genes is also presented. Inflammation involves a complex set of interactions triggered in response to tissue injury (16, 17). Once initiated, a cascade of cellular infiltrations and molecular releases occur that ultimately results in local cellular proliferation at the site of intlammation to repair damaged tissue. Normal inflammation is self-limiting; pro-inflammatory cytokines that promote proliferation and repair are closely followed by the production of anti­inflammatory cytokines that facilitate the resolution of the inflammation. Some of the inflammatory mediators involved in the proliferation and repair response include arachidonic acid (AA) metabolites, cytokines, chemokines, free radicals, reactive oxygen species, and adhesion molecules. Chronic exposure to these mediators leads to prolonged 3 cell proliferation and eventually to mutagenesis, oncogene activation, tumor suppressor inactivation, and angiogenesis. 1.1.2 Tnflamlnation and Cancer Inflammation and cancer share some basic characteristics. In fact, it has been proposed that tumors act as "wounds that do not heal" (18). There are many examples of inflammatory cells assembling in and around a tumor microenvironment (19). For a long time, inflammatory conditions surrounding the tumor microenvironment were thought to be an immune response to the cancer and likely a positive sign that the body was - responding to the tumor with its first line of defense. However, that view has changed after numerous studies illustrated that the presence of inflammatory mediators in and around the tumor microenvironment were actually promoting tumor growth rather than mounting an effective immune response (20-22). - Tumor cells can produce various cytokines and chemokines that attract leukocytes. These inflammatory cells subsequently produce cytokines, chemokines, and other metabolites that stimulate additional tumor cell proliferation (23). The inflammatory milieu is teeming with AA metabolites, pro-inflammatory cytokines, chemokines, reactive oxygen species (ROS), adhesion molecules, and proteases which aid and encourage tumor growth (Figure 1.1). At the molecular level, free radicals, aldehydes, and other reactive metabolites produced during chronic inflammation can induce harmful gene mutations and yield unwanted posttranslational modifications of proteins. Cytokines, growth factors, and transcription factors control the regulation of genes and induce the expression of proteins involved in both inflammation and cancer. 4 Figure 1.1 Chronic Inflammation Predisoposes to Cancer. This diagram represents a proposed model of chronic inflammation in the development of cancer. Chronic inflammation leads to an environment of proliferation and oxidative damage; constant exposure to this environment promotes oncogenic mutations leading to tumorigenesis. Adapted from Am 1 Physiol Gastrointest Liver Physiol2004; luI; 287, G7-17 (24). Chronic Inflammation Predisposes to Cancer INormal Tissuel Chronic Inflammatory StirYluli f----------. Injury, Infection Inflammatory Cells 1 COX-2, LOX /~.-------- Inflammatory Oxidative Stress Mediators ROS,RNS Reactive Lipids I Inflamed Tissue I .. 5 Eicosanoids IDamag ed Tissuel DNA, Protein, Lipid Adduct -----------.­Improper Signaling Genetic/Epigenetic Alterations Dysplasia I Additional Genetic Changes If- ----~ -_ -_ -+_-_1 __ -----, Cancer 6 For example, inducible inflammatory enzymes such as nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), and 5-Lipoxygenase (5-LOX) are controlled by inflammation-mediated growth factors and transcription factors. These inducible enzymes in turn directly influence eicosanoid levels and concentrations of reactive species (RS), such as ROS, reactive nitrogen species (RNS) and reactive oxygen lipid species (ROLS). Some DNA and protein damage to the host is inevitable with host­defense processes that evolved to eradicate pathogens and some risk of cancer is inevitable if DNA synthesis, cell proliferation, growth and angiogenesis favor selection of a cell with a mutant genome. The combined result is an environment that over time promotes DNA and protein damage, DNA synthesis, cellular proliferation, survival, and angiogenesis (23). 1.1.3 Eicosanoids Eicosanoids (derived from the Greek eicosa meamng twenty; referring to twenty carbon fatty acid derivatives) are oxygenated and biologically active derivatives of the 20-carbon essential fatty acid AA. These include prostaglandins (PG), thromboxanes (Tx), prostacyclin (PGl), and leukotrienes (L T). Eicosanoids are not stored in cells but rather are newly synthesized when needed. The AA precursor is stored in cells primarily in an esterified form at the 2-acyl position of phospholipids in all mammalian outer and inner membranes. AA must be hydrolyzed from the membrane prior to utilization tor eicosanoid synthesis. In response to perturbation, injury, or other inflammatory stimuli, AA is released from cell membranes by the action of phospholipase A2 (PLA2) and is subsequently oxidized by AA-metabolizing enzymes, such as COX or LOX. Ultimately, the different types of eicosanoid metabolites formed following AA release depend on the 7 kinds and combinations of converting enzymes present in the stinlulated cell or tissue. Eicosanoids are formed in all mammalian tissues and are generally thought to playa pro­inflammatory role. They are also implicated in cancer initiation and progression (25). A greater understanding of these and other proposed mechanisms associated with inflammation-induced carcinogenesis will yield insights into potential targets for therapeutic prevention and treatment of cancers. 1.2 Cyclooxygenase Enzymes Cyclooxygenase (COX; also known as Prostaglandin G/H synthase (PGHS)) is an enzyme that catalyzes the committed step in the conversion of AA to PG metabolites. In the 1930s, prostaglandins were first discovered as potent bioactive lipid messengers that could be extracted from semen, prostate, and seminal vesicles (hence the name prostaglandins, or fronl prostate glands) (26). in the 1960s, the structures of prostaglandins were elucidated and AA was identified as their biosynthetic precursor fatty acid (27). It was not until the mid-1970s that the enzyme responsible for catalyzing the cyclooxygenation reaction of AA was purified from sheep seminal vesicles (28). This purified enzyme, COX, was found to be approximately 67,000 daltons and to contain both cyclooxygenase and peroxidase activities. The existence of multiple isoforms of COX was speculated early on. It was not until 1991, however, that the second isoform COX-2 was identified (29). Interestingly, in 1971, it was discovered that aspirin, indomethacin, and other popular nonsteroidal anti-inflammatory drugs (NSAIDs), were inhibitors of the enzyme responsible for the biosynthesis of prostaglandins (i.e. COX) (30). This discovery elucidated the mechanism of action of salicylates, an important 8 class of dnlgs that had been in use for over 100 years, and paved the way for further advances in COX, prostaglandin, and NSAID research and discovery. 1.2.1 Cyclooxygenase Structure There are two major isozymes of COX found in humans: COX-l and COX-2 (31). The primary structure of these enzymes is similar. COX-l is comprised of 576 amino acids and COX-2 is comprised of 587 amino acids; there is a 600/0 sequence identity between the enzymes. COX-2 has a unique 18 amino acid insert in the C-terminal end which is speculated to regulate the rapid degradation of COX-2. COX-l has additional amino acids at the N-terminus not found in COX-2 that comprise part of a signal peptide. COX-I and COX-2 are homodimers both functionally and structurally, although it is not known why dimerization is necessary for catalysis. Each monomer of COX consists of three structural domains: an epidermal growth factor (EGF) domain at the N-terminus, a membrane binding domain (MBD), and a large catalytic domain at the C-terminus (32). The EGF domain forms a portion of the dimer interface and is necessary for proper folding. The enzymes are targeted to the luminal surfaces of the endoplasmic reticulum and the inner and outer membranes of the nuclear envelop via the MBO. The large globular catalytic domain contains both the cyclooxygenase and peroxidase active sites of COX. The cyclooxygenase active site is a long, hydrophobic channel that originates at the MBO and extends into the globular catalytic domain (33). This allows AA and O2 to enter the mouth of the channel directly from the lipid bilayer. The hydrophobic channel of COX-I and COX-2 ditTcr at one important residue: lIe at position 523 in COX-I and Val at position 523 in COX-2. The lIe:Val exchange at this position makes the active site of COX-2 about 20% larger than that of COX-I. This size ditTerence has becn exploited 9 to make compounds that are COX-2 specific inhibitors (34). The peroxidase active site is also found in the globular catalytic domain of COX at a site furthest from the membrane. This site contains a heme group bound by an iron-histidine bond. 1.2.2 COX Function and Catalysis COX-l and COX-2 have very similar active site structures, catalytic mechanisms, products, and kinetics (31). Both COX isozymes catalyze the oxidation of AA to PGH2• The catalysis of AA into PGH2 occurs by two separate activities of COX: cyelooxygenase and peroxidase. The cyelooxygenase activity of COX catalyzes the addition of one molecule of O2 to carbon 11 of AA. There is a subsequent rearrangement in which the molecule of O2 bridges carbon 9 and carbon 11 to form a cyelic endoperoxide and a new carbon-carbon bond fonns between carbon 8 and carbon 12 to yield a cyelopentane ring. This reaction occurs via a free radical mechanism involving a tyrosine radical (35). The cyelooxygenase active site also catalyzes the addition of a second molecule of O2 which yields a hydroperoxyl group at carbon 15; this metabolite is known as PGG2. The peroxidase activity of COX reduces the hydroperoxyl group at carbon 15 to a hydroxyl via a two electron transfer at a heme active site to form PGH2. These two reactions occur at distinct sites. The peroxidase reaction occurs at a heme­containing active site located near the protein surface; the cyelooxygenase reaction occurs in the hydrophobic channel located ncar the core of the enzyme. It is thought that following PGG2 synthesis at the cyclooxygenase site, PGG2 exits the mouth of the channel and diffuses to the peroxidase site on the same monomer or opposite monomer of the COX homodimer. Despite their location differences, activity at the cyelooxygenase site is dependent on activity at the peroxidase site. Likely, the peroxidase activity of 10 COX provides the oxidant to form the tyrosine radical necessary for AA oxidation. Interestingly, the converse does not appear to be true as the peroxidase active site can function without activity at the cyclooxygenase site. 1.2.3 COX Regulation Regardless of the structural similarity of the COX isozymes and the fact that they catalyze identical reactions, there are important differences that necessitate having both COX-l and COX-2. An obvious difference between COX-l and COX-2 is gene regulation (33). COX-I is predominantly a constitutively-expressed enzyme and is found in nearly all tissues throughout the body. COX-l is therefore responsible for certain homeostatic functions, such as maintaining normal gastric mucosa, influencing renal blood flow, and aiding in blood clotting by abetting platelet aggregation (36). In contrast, COX-2 is an inducible enzyn1e expressed in response to pathological or physiological stresses. The COX-2 gene is particularly responsive to, and most commonly elevated by, growth factors and mediators of inflammation such as IL-I, TNFa, NF-KB, and lipopolysaccaride. Thus, COX-2 primarily provides the PGs that mediate pain and fever, and support the inflammatory process (37). In general, this simpli fication of COX-I being involved in house-keeping functions and COX-2 mediating pain and inflammation works; however, there are some data that suggest these isozyme functions are not quite so unambiguous. Despite the significant difference in gene regulation, COX-I and COX-2 have very similar cyclooxygenase and peroxidase specific activities. This raises the question of how the isozyn1es are regulated and distinguished when expressed in the same cell. The subcellular localization of the isozymes is very similar, although COX-2 is slightly more 11 concentrated in the nuclear envelope than COX-l (38). This difference in location might provide one mechanism of selectivity between the activity of COX-l and COX-2. Another mechanism that may help distinguish the isozymes is the fact that COX-l exhibits negative allosterisl11 at subl11icromolar concentrations of AA well below the Km whereas COX-2 does not (39). At low concentrations of AA (S; 1 ~M), COX-2 is nearly four times n10re efficient at PG synthesis than COX-I. This likely plays a role during the initial stages of AA release by giving COX-2 a metabolic advantage over COX-I. 1.2.4 COX Metabolites COX catalyzes the formation of PGH2 from AA (Figure 1.2). PGH2 is subsequently converted into a class of metabolites termed prostanoid'i which includes PGs, prostacyclin (PGb), and thromboxanes (Tx). The synthesis of prostanoids is accomplished in a stepwise manner by a complex of microsomal or cytosolic synthases and isomerases. The coupling of PGI-b synthesis to further metabolism by downstream enzymes is intricately orchestrated in a cell dependent manner (40). For example, thromboxane synthase is mainly expressed in platelets and macrophages to form TxA2, a potent vasoconstrictor and platelet aggregator. Conversely, prostacyclin synthase is primarily found in endothelial cells to form prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. Prostaglandin synthases 0, E, and F form PGD2, PGE2, and PGF 2(1, respectively~ these enzymes are also regulated in a cell specific manner. Prostaglandin synthase F is primarily found in the uterus whereas prostaglandin synthase o is found in mast cells. Prostaglandin synthase E is found in almost all cells, demonstrating the important role PGE2 plays in both homeostatic and inflammatory functions. PGD and PGE can undergo spontaneous or enzymatic dehydration to form 12 PGs of the J- and A-series (41), also called cyclopentenone PGs (cyPGs). These prostaglandins contain an clectrophilic, cyclopentenone ring structure that is important in both the inflammatory and tumorigenic actions of the cyPGs. Although PGs playa substantial role in homeostatic functions in the body, the focus here is the role PGs play in prolTIoting inflammation. During an inflammatory response, both the level and profile of prostanoid production change dramatically (42). PGs are found at low levels in all cells but increase rapidly following inflammatory stimuli prior to leukocyte infiltration. A further increase in prostanoids occurs with the infiltration of immune cells (43). Early events in the inflammatory response such as increased vasodilation and vascular permeability are mediated by COX-2 generated PGE2 and prostacylin, both of which are potent vasodilators. Subsequently, the prostanoids can also affect immune cell functions via binding to G protein-coupled cell surface receptors. The actions of PGF2u, PGh, and TxA2 are mediated by individual receptors: FP, IP, and TP, respectively (44). PGD2 and PGE2 have multiple receptors: DP 1 and DP2, EP I-EP4. Prostanoid receptors couple to a range of intracellular signaling pathways that mediate the effects of receptor activation on leukocyte function. Reversible binding of prostanoids to speci fic receptors cause increases or decreases in intracellular cAMP levels, resulting in the activation or inhibition of various intlammatory cells and changes in the secretion of cytokines and chemokines. Additionally, certain prostanoid receptors couple to mobilization of intracellular calcium, which can activate immune cells and phospholipases. Cyclopentenone PGs have been reported to have both pro- and anti­inllammatory effects. At low concentrations, cyPGs aid the inflammatory response by 13 Figure 1.2 Cyclooxygenase Pathway. Schematic represents formation of prostaglandins via oxidation of Arachidonic Acid (AA). AA is oxidized by COX-lor COX-2 to PGH2, which is subscqucntly metabolized to other PGs by specific PG synthases. The solid arrows represent enzymatic pathways and the dotted arrows represent nonenzymatic or spontaneolls dehydration of PGs. HO o o Cyclooxygenase Pathway Arachidonic Acid .,' 1 Cyclooxygenase ."~COOH OOH PGG2 " 1 Peroxidase /~COOH OH PGH2 PG Synthases .·""~COOH '''. T/'-.., /'--.. ... " ~- ........... COOH OH PGJ2 HO OH PGF2a o HO o 14 COX-1/2 ,,". /'-.., /'--.. .. ,' ~- .......",. COOH - OH PGE2 '''. T /'-.., /'--.. ... " ~- ........... COOH 15 inducing production of reactive oxygen specIes, activating the extracellular signal­regulated kinase 1/2, and in turn increasing proinflammatory cytokine expression (45). The cyPGs also inhibit inflammation by activating nuclear PPARr receptors, which decrease cytokine release, (46) and inhibit NF-KB translocation to the nucleus by covalently modifying and inhibiting IKB Kinase (47). Although PGs are generally thought to be pro-inflammatory, they play an important role in regulating resolution as well. At a molecular level, PGE2 primes cells for the subsequent anti-inflammatory pathways by amplifying lipoxins, a distinct class of LOX-derived eicosanoids with potent anti-inflammatory activity (48). 1.2.5 COX and Cancer Prostanoids have long since been thought to playa role in the pathogenesis of cancers. Early studies found that growth factors, tumor promoters, and oncogenes induce prostanoid synthesis and that metabolism of AA via the COX pathway is enhanced in human tmnors compared to the nontumorigenic counterparts. This finding eventually led to studies examining the expression of COX-I and COX-2 in tumor tissues. Early studies demonstrated increased COX-2 expression in greater than 800/0 of colorectal cancer tissues compared to normal adjacent tissues (49). Notably, COX-2 expression was not limited to inflammatory cells but was present in epithelial cells as well (50). The over­expression of COX-2 has been shown in tumors of the pancreas, breast, skin, gastrointestinal tract, bladder, lung, prostate, head and neck, and others (51). These data led to the hypothesis that COX-2 was playing a role in cancer development and progression. Genetic and pharmacological inhibition studies have corroborated the fact that there is a causal role of COX-2 in cancer developnlent. 16 It is widely accepted that COX-2 plays a role in tumor initiation and progression; however, the mechanisms involved in this process are not fully understood. As expected, the overexpression of COX-2 in premalignant and malignant tumors is consistently associated with an increase of COX-2 derived PGs. It is also accepted that the increase in PGs is playing a tumorigenic role. There are numerous mechanisms proposed that link PGs to cancer, which are discussed below. It is likely that the combination of these and others result in the promotion of tumorigenesis. Studies demonstrate that PGE2, PGF2a, and PGD2 signal via EP, FP, and DP receptors, respectively, to stimulate proliferation, survival, invasion and angiogenesis (44). Crosstalk of PGE2 and PGF2a with different growth factor receptor-induced signaling cascades stimulates cell proliferation. For example, PGE2 has been shown to activate epidermal growth factor receptor (EGFR) by increasing EGF or through binding to the EP receptors (52, 53). PGE2 can also propagate Vascular Endothelial Growth Factor (VEGF) signaling via the induction of VEGF expression (54). PGE2 has additionally been shown to promote cell proliferation by activating several important oncogenic pathways including Ras-MAPK, PT3K-Akt, and Wnt signaling (55-57). PGE2 can inhibit apoptosis by up-regulating Bcl-2 expression (58) or stimulating NF-KB (59). Pro­angiogenic effects of aberrant COX-2 expression are mediated through PGh, PGF2{1, and particularly PGE2, which increase vasodilation and vascular permeability via receptor binding (60). Another important aspect to consider is the carcinogenic potential of reactive chemical species generated via the COX pathway (Figure 1.3). During prostaglandin biosynthesis, COX-2 generates ROS that may playa direct or an indirect role as a 17 Figure 1.3 Peroxyl Radical and Aldehyde Formation during Peroxidation of Arachidonic Acid. Both COX-2 and LOX enzymes catalyze the peroxidation of AA via a radical mechanism. In this process, a peroxyl radical is fonned which can react with DNA, proteins, and lipids to cause oxidative damage. Decomposition of the peroxyl radical forms reactive aldehyde species, which can cause alkylation adducts to DNA and proteins. 18 Peroxyl Radical and Aldehyde Formation during Peroxidation of Arachidonic Acid t--H C~COOH 1 Diene Rearrangement I [C?21 Vo. 0/ COOH.--_> Oxidative Damage Peroxyl Radical COOH j Decomposition o ~COOH .---r--.... Alkylation Damage Reactive Aldehydes 19 carcinogen (61). The COX-2 generated ROS can result in oxidative damage by directly oxidizing proteins and DNA. Additionally, ROS can oxidize lipids which damage proteins and DNA via unwanted covalent lipid modifications (62). Catalysis of AA by COX produces a peroxyl radical intermediate within the lipid membrane. Peroxyl radicals are amongst the n10re stable oxy radicals, which allows for greater diffusion from the site of formation. Diffusion of these intermediates from the enzynle active site can initiate lipid peroxidation reactions within the nuclear membrane where thcy can decompose to reactive aldehydes or epoxides (Figure 1.3) (63). These reactive byproducts may act as carcinogens by reacting with nucleophilic residues on proteins or DNA (64). Another carcinogenic byproduct of prostaglandin biosythnesis is the nonenyzmatic or enzymatic (via thromboxane synthase) breakdown of PGH to malondialdehyde (MDA) (65). MDA binds to DNA and produces base-pair substitutions and frameshift mutations. MDA can also covalently modify and inactivate important cellular proteins (66). Dehydration products of various prostaglandins, such as PGA2 and PGh, are reactive metabolites with an electrophilic a,p-unsaturated ketone. These electrophilic metabolites can also calise adducts to nucleophilic residues on proteins via a Michael addition (Figure 1.4), thereby promoting a tumorigenic environment (67, 68). 1.3 Lipoxygenase Enzymes LOXs are a class of lipid dioxygenases which play important roles in inflammation, asthma, and several diseases including cancer. LOXs arc found widely in animals, 20 Figure 1.4 Michael Addition. Schematic represents a Michael Addition of a cysteine residue on an enzyme with PGA. This is the mechanism by which electrophilic metabolites covalently modify nucleophilic residues. First, a nucleophilic, ionized sulfur atom on cysteine attacks the ~-carbon of the cyclopentenone moity, resulting in a shift in electrons towards the carboxyl group and a covalent bond between the sulfur and the ~­carbon. Next, the ionized oxygen rcfonns the carbonyl and a hydrogen is accepted at the a-carbon to form the final adduct. 21 Michael Addition ~ Enzyme S OH plants, and fungi. A pro-inflammation mammalian LOX was first described in 1979 (67). This LOX found in leukocytes converted AA into a hydroxylated product (69). The products resulting from LOX activity were callcd leukotrienes, from the immune cells they were discovered in (leukocytes) and from a common structural feature of the products containing three conjugated double bonds (tricne) (70). There are several isoforms of LOX which are categorized with respect to their positional speci ficity of AA oxygenation (i.e., 5-, 8-, 12-, 15-LOX) and if necessary the stereochemistry (S or R) of O2 insertion. 1.3.1 Lipoxygenase Structure LOX proteins are comprised of a single polypeptide chain with a molecular mass of 75,000-80,000 daltons (71). They have a small N-terminal p-barrel domain and a large catalytic C-terminal domain. The catalytic domain contains a well-conserved active site comprising of a single atom of nonheme iron. The active site iron is coordinated to three conserved histidine residues and to the carboxy group of a conserved isoleucine (72). LOXs are inactive while the iron is in the ferrous form and requires oxidation to the active ferric iron. It is not entirely clear how the substrate gains access to the iron active site but likely occurs via a ncarby channel that opens onto the top surface of the protein. 1.3.2 Lipoxygenase Function and Catalysis LOXs catalyze the oxidation of AA and other free fatty acid substrates to form hydroperoxy-eicosatetraenoie acids (HpETEs). This reaction occurs via a multi-step process at the iron catalytic site. First, the stereospecific removal of the pro-S hydrogen from a I, 5-cis, cis-pentadiene structure occurs, leaving a pentadienyl radical intermediate 23 (73). Following this step, a radical rearrangement occurs resulting in diene conjugation and a carbon radical at the position of oxygenation of the LOX (i.e. C-5 for 5-LOX). Next, a stereospecific insertion of a molecule of oxygen occurs at the carbon radical resulting in a hydroperoxyl radical. Finally, the hydroperoxyl radical is reduced to the corresponding anion and a proton is accepted to form the hydroperoxl group. The one electron transitions are catalyzed by the iron in the catalytic center of the enzyme; the ferric iron (III) is reduced to ferrous iron (II) and back to ferric iron (III) during the course of the AA oxygenation by LOX. The hydroperoxy group can be reduced to the corresponding hydroxyl to form hydroxyl-eicosatetraenoic acids (HETEs) by phospholipid hydroperoxidc glutathione peroxidase (PHGPx or GPx4) (74). 5-LOX is unique in that this LOX possesses two distinct enzymatic activities. 5-LOX catalyzes the oxygenation of AA to 5-HpETE. 5-HpETE can be reduced to 5-HETE by GPx4 or 5-LOX can catalyze the epoxidation of 5-HpETE to an unstable 5,6-epoxide known as Leukotriene A4 (L T A4) (75). L T A4 is converted into biologically active leukotrienes (L T) by multiple pathways depending on the cellular location. Because of the role 5-LOX plays in synthesizing L Ts and the importance of 5-LOX in int1ammatory reactions and cancer, 5-LOX will be the LOX isozyme of prinlary focus herein. 1.3.3 Lipoxygenasc Regulation There arc five active human LOXs: 5-LOX, 12(S)-LOX, 12(R)-LOX, 15-LOX-I, and 15-LOX-2. Expression of these enzymes largely depends on the cell type (76, 77). LOXs are primarily found in immune cells. For example, platclets have 12(S)-LOX; leukocytes contain both 5-LOX and 12(S)-LOX; epidermal cells contain a subgroup of LOXs including 12(S)-LOX, 12(R)-LOX, 15-LOX-I, and 15-LOX-2; eosinophils, mast 24 cells, polymorphonuclear leukocytes, and n10nocytes express 5-LOX. Besides cell­dependent expression, these enzymes are regulated by having different kinetics and specificities towards their substrates. For example, 15-LOX-l prefers linoleic acid as a substrate whereas 15-LOX-2 prefers AA (78). 5-LOX is regulated by four different mechanisms. First, calcium is a key regulator for both 5-LOX oxygenation activity and L TA4 synthesis (79). 5-LOX reversibly binds free calcium which stimulates activity (80), presumably by increasing the hydrophobicity and subsequently men1brane association. Second, ATP can also bind to and activate 5-LOX (81). Third, Jipoxygenase activity of the 5-LOX requires the oxygenation of the ferrous iron of the active site to the ferric form. Thus, lipid peroxidases, including the HpETE products of other LOXs, can accomplish this iron oxygenation and subsequent activation of 5-LOX (82). Finally, 5-LOX is regulated the 5-LOX activating protein (FLAP) (83). Following various stimuli, 5-LOX translocates to the membrane and binds to FLAP. FLAP acts as an AA carrier to 5-LOX, thereby playing a crucial role in the synthesis of LTs. 1.3.4 Lipoxygenase Metabolites Regioisomeric LOXs catalyze the oxygenation of AA to the corresponding HpETE, which is subsequently reduced to the analogous HETE. These HETE products play important roles in many biological processes. 15(S)-HETE is implicated in cell differentiation, int1ammation, and asthma (84). 12(S)-HETE promotes chemotaxis (85) and synthesis of heat shock protein in leukocytes (86). Jt can also cause constriction of the blood vessels and inactivation of prostacyclin synthase (87). The other enantiol11cr, 12(R)-HETE is a chemoattractant for polymorphonuclear leukocytes (88). 5(S)-HpETE 25 is an important metabolite in respect to it being a precursor to L T formation; however, there arc some biological activities attributed to the reduction of 5(S)-HpETE to 5(S)­HETE, such as activation of monocytes and neutrophils (89). In two consecutive reactions, 5-LOX converts AA to L T A4; this highly unstable epoxide intermediate can undergo enzymatic hydrolysis to form L TB4 or be conjugated to glutathione to form LTC4 (Figure 1.5) (90). L TC4 can be further metabolized to L TD4 or L TE4; collectively these metabolites are referred to as the cysteinyl-containing L Ts (cys­L T). L Ts possess a wide range of biological activities mediated primarily upon binding to specific G-protein-coupled, cell surface receptors. LTB4 is a very potent chemoattractant for neutrophils and recruits inflammatory cells to the site of injury (91). The cys-L Ts are potent constrictors of smooth muscle; this is particularly important in the airways where they elicit bronehoconstriction (92). In the microcirculation, they evoke constriction of arterioles and increase the vascular penneability, leading to invasion of the plasma. Fatty acids have long been implicated in the process of carcinogenesis. Tumorigenesis studies demonstrated that polyunsaturated fatty acids must undergo oxidative metabolism to enhance tumorigenesis (93), suggesting a role for both LOXs and COXs. Various LOX isozymes, including and 12-LOX, and their respective products have been linked to tumorigenesis in experimental Inodels; modulation of these LOX metabolites has anti-tumorigenic effects in these models (94). Interestingly, 15- 26 The 5-Lipoxygenase Pathway Arachidonic Acid 5-LOX 1 OOH 5-HpETE OH COOH eOOH 5-HETE COOH OH cyLTs Figure 1.5 5-Lipoxygenase Pathway. This schematic represents AA metabolism by 5- LOX resulting in 5-1IETE or L T products. 27 LOXs, which also metabolize AA, do not appear to playa major role in tumorigenesis and may, conversely, have an anti-tumorigenic role (95). Emerging in recent years is the role of 5-LOX in cancer. 5-LOX is expressed by a broad variety of cancer cells including lung, breast, pancreas, prostate, mesothelium, brain, and others (96). For human pancreatic cancers and mesotheliomas, 5-LOX is expressed at high levels in the cancerous tissues but is found at very low levels in the corresponding normal tissue (97, 98). Besides tumor cells, strong 5-LOX expression was found in infiltrating macrophages and microglial cells (99). It appears, then, that increased 5-LOX expression corresponds with neoplastic transformation in certain tissues. Several 5-LOX metabolites have been implicated in the progression of cancer. 5-HETE can inhibit apoptosis and support cell proliferation by activating the MEKJERK and PI3KJ Akt pathways (100, 101). 5-HETE can contribute to angiogenesis by activating MMP2 and VEGF generation (102). L TB4 can inhibit apoptosis by inhibiting cytochronle c release and caspase activation (103). Adding to the tumorigenic threat, L TB4 plays a role in promoting cell proliferation by binding to its receptor and stimulating concurrent activation of the MEK/ERK and PI3KI AKT pathways (104). L TB4 is implicated in invasion and metastasis, possibly by increasing trans-endothelial binding and migration ( I 05). Several lines of evidence indicate that the 12(S)-LOX product 12(S)-HETE contributes to carcinogenesis. Platelet-type 12(S)-LOX is up-regulated in several human cancers, such as prostate, and correlates with the tumor grade and stage. 12(S)-HETE has been shown to promote tumorigenesis by up-regulating adhesion molecules and increasing the adhesion of tumor cells to microvessel endothelium (106). 12(S)-HETE 28 can also enhance cell migration and promote tumor spreading by activating protein kinase C (PKC) (107). LOXs also contribute to the carcinogenesis process by increasing ROS and ROLS (62). LOX pathways catalyze the formation of HpETEs, which are by definition a ROLS. Additionally, peroxidative cleavage of HpETEs results in an increase in ROS. ROS and ROLS can regulate the activities of several kinases, phosphatases, transcription factors, cell death machinery, and proteins such as COX-2 and iNOS (108). Another important reactive byproduct of this pathway is the lipid peroxidation decomposition product 4-hydroxynonenal (4HNE) (109). 4HNE, an ROLS with an a, ~-unsaturated hydroxyalkenal, is a potent carcinogen. Prolonged ROS generation and exposure leads to deleterious effects such as protein and DNA oxidation (Figure 1.6). Prolonged ROLS exposure can lead to unwanted protein and DNA adducts. 1.4 Tumor Suppressors There are several different mechanisms proposed to link eicosanoids to intlammation and cancer discussed above. In the subsequent chapters, a novel mechanislTI is presented linking eicosanoid biosynthesis by COX-2 and 5-LOX to inactivation of tumor suppressors. It is necessary, then, to spend a little time discussing tumor suppressor genes and their mechanisms of inactivation. Genes involved in the etiology of cancer are broadly defined into two categories: oncogenes and tumor suppressors. Oncogenes promote cancer whereas tumor suppressors protect from cancer. In 1971, Alfred Knudson Jr. published a seminal work on tumor suppressors, hypothesizing that tumor suppressors are recessive genes which must have two hits for inactivation (110). This work was done studying retinoblastoma 29 Figure 1.6 Oxidation of Cysteine Residues. Schematic represents oxidation of ionized cysteine by ROS. Path 1 represents oxidation to a sulfenie acid. The sulfenie acid can either form a sulfenamide (2) or a disulfide (3), of which both species can be converted back to cysteine. Overoxidation results in formation of a sulfinie (4) or sulfonic (5) acid. These species arc not reduced by cellular reductants and therefore considered permanent oxidative modifications. Oxidatiol1 of Cysteine Residues Cys~S / ROs1 Cys~S N-R Cys~S-OH ROS 4 o ~ II CyS/ 'S-OH ROS 5 o ~ II CyS/ "S-OH II o 30 31 and indeed this tumor suppressor fits the two hit model. For many years now, the two hit model has been a hallmark of tumor suppressor genes. One important issue is what defines a hit. For Knudson, a hit was a permanent genetic inactivating event. This includes mutations, deletions, or loss of heterozygosity (LOH) (111). Mutations can be inherited or acquired in a tumor suppressor gene. These mutations can be point, null, missense, or nonsense mutations. Ultin1ately, these mutations result in inactivation of the tumor suppressor gene or protein encoded. Deletions are genetic aberrations in which part of the chromosome or sequence of DNA is n1issing. This can occur during chron10somal crossing in meiosis. Deletions can result in frameshifts or badly truncated proteins. LOH occurs when there is a complete loss of one of these alleles. Any combination of these mechanisms inactivating both alleles would satisfy the two hit hypothesis. As with most things, there are exceptions to the rule (112). There are numerous tumor suppressors that do not appear to satisfy the two hit model. For example, several known tumor suppressors demonstrate haploinsufficiency, or abrogated function due to loss or inactivation of a single allele. For example, LKB 1 (113) and PTEN (114) tumor suppressors, which are discussed in the following chapters, are haploinsufficient tumor suppressors. These tumor suppressors express wild-type protein from one intact allele, albeit at a lower level than in normal tissues, yet lack their tumor suppressor function. Several tumor suppressors display dominant negative isofonns in which missense or point mutations yield a nonfunctional protein that impedes the wild-type protein function. A TM is an example of a tumor suppressor that exhibits a dominant negative isoform. A TM is a protein that senses genomic stress and the most common type of mutation of 32 A TM is a missense mutation (115). These missence mutants of ATM are non-functional, yet they interfere with wild-type ATM function (116). Alternately, some tumor suppressors display gain of function isoforms where mutation results in a functional protein with novel tumor promoting functions. Although this by definition is an oncogenic mutation, several tumor suppressors have been shown to promote cancer by this gain of new function rather than loss of function. A classic example of gain-of­function tumor suppressors is p53. Mutations that result in an altered conformation of p53 yield a protein that interferes with normal spindle checkpoint regulation (117). These are a few examples of tumor suppressors that do not appear to meet Knudson's two hit model. A relevant Issue to consider is the definition a hit. Traditionally considered an irreversible genetic alteration, there are other mechanisms that result in inactivation of a tumor suppressor. For example, gene silencing by CpG island hypermethylation can result in decreased expression of the tunlor suppressor gene and protein. Tazarotene induced gene 1 (TIG 1) is silenced by methylation in human cancers (118). This epigenetic process differs from mutational disruption of genes as methylation is provisionally a reversible process. Chromosome remodeling is also a key regulator of gene transcription ( 119). The chromosome can be altered structurally by posttranslational histone modifications or by ATP-dependent chromosome remodeling. Deregulation of chromosome-remodeling activity can lead to cancer, perhaps due in part to altered gene transcription of a tunlor suppressor. Another mechanism controlling gene expression is microRNA (120). MicroRNA are transcribed from DNA but are not translated into protein. MicroRNA anneal to a complementary messenger RNA and 33 inhibit protein translation; in this way, microRNA can bind to tumor suppressor mRNA and prevent protein expression. MicroRNA has been shown to have oncogenic function by inhibiting the expression of LATS2 tumor suppressor (121). Additionally, DNA viral oncoproteins can bind to tumor suppressor proteins, such as retinoblastoma and p53 (122). This interaction disrupts the normal function of the tumor suppressor. While not traditional genetic hits, all these mechanisms prevent the normal activity of the tumor suppressor and are thus functionally analogous to a classical hit. Ultimately, it seems as if a more relaxed definition of a hit is any mechanism that results in the loss of the intended function of the gene product. In this dissertation work, I have investigated inactivation of tumor suppressors at the protein level. I proposed that ROS and ROLS generated during eicosanoid biosynthesis by COX and LOX enzymes caused unwanted chemical modifications of tumor suppressor proteins. These modifications are analogous to a genetic or epigenetic hit as they prevent the normal function of the tumor suppressors. I investigated the modification of two tumor suppressors: LKB 1 and PTEN. I found that ROLS covalently modified LKB 1 and that ROS oxidized PTEN. These modifications occur on key residues of the proteins and result in the loss of function of the tumor suppressors, thereby allowing propagation of signals through oncogenic pathways. As mentioned, both LKB 1 and PTEN are haploinsufficient tumor suppressors. Inactivation of LKB 1 and PTEN at the protein level by this mechanism may reconcile these tumor suppressors with Knudson's two hit hypothesis under certain conditions, sueh as cancers related to chronic intlammation or increased expression of COX-2 and LOX. 34 1.5 References 1. Trichopoulos, D. 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Mullally, Frank A. Fitzpatrick Published as: Wagner, T.M., Mullally, J.E., and Fitzpatrick, F.A. (2006) Reactive lipid species from cyclooxygenase-2 inactivate tumor suppressor LKB I/STK II: cyclopentenone prostaglandins and 4-hydroxy-2-nonenal covalently modify and inhibit the AMP-kinase kinase that modulates cellular energy homeostasis and protein translation. Journal of Biological Chemistry 281, 2598-2604 Reprinted with permission 47 Reactive lipid Species from Cyclooxygenase-2 Inactivate Tumor Suppressor LKB1/STK11 CYCLOPENTENONE PROSTAGLANDINS AND 4-HYDROXY-2·NONENAL COVALENTL Y MODIFY AND INHIBIT THE AMp·KINASE KINASE THA T MODULATES CELLULAR ENERGY HOMEOSTASIS AND PROTEIN TRANSLA TlON* Tracy M. Wagner·, James E. Mullally, and F.A. Fitzpatrick From the Department of Medicinal Chemistry, University of Utah Huntsman Cancer Institute, Salt Lake City, Utah 84112 LKBI. a IIniqu(' serin~lthrt'onin(' kim,se tumor Sl'PI'r('ssor. mod­ul. lles anabolic and catalmlic hOI1l~ostasis. cdl prolif~ralion. ulld orgal! polarity- Chcmkally I'('actiw lil'ids, !'.g. cydop('ntenone 1'1'08- laglandins.li.lrnu,c\ a c:<.,,"il"·ntadduf"l wilh [.Kill in MCF-i ,mel RKO fells. Site· directed mutagenesb implicated Cp,'HI inlhc LKIH <ltti· vat;o" loop as th" ",sid",· modifi"d. Nnt"hly. FRK, INK. and ,\KT serine!lhf(~Clninc kinases with Jeudnl~ ur mclhionhu~, inslt~acJ of cp~ .. !('int'. in thd,. af\i,·ation lOOp did nOI (OI'Ill a (ovalt:'lIllil,id adduct. ". Hydroxy"::!. nnncnul, 4~oxn·· 2··nuncnaI1 llnd crdnpenh~nunc pros· taglandin A and J, whkh all (ontain <l',/J'lIl1saturalcd carbunyls, inhibited the A;>'lP-kina~e kinas(' "CliYit)' of ("lIl1lar LKllI.ln till'll, this attenuated signals throughollt the LKBI - AMI' kin~sc palh W'I)' and disl'uptccl its restraint of ribosomal S6 ki !lase;,. The c1cctr<l­phillc If· carbon in these lipids appears to be critical for inhibition be,auso; unrc;\tth'c lipid., "'Ii- l'GL\1' I'G[., [,GI',,'" and '1',,11,. did not inhibit LKBI activity (I' > 0.05). Ectopic expression of cydoox~'· gcna.<;e··2 undendogt:'nolls biosynthesis ofcicosanoids also inhibited l.l;(IH acth·ity in Mer-7 ,dis. Our n:suhs suggested a moit'(ular mechanislll wh('rchy chronk inflammation or oxid"tiw stress 01,,)' ,onfer risl( for hypertrophic or neoplastic diseases. "\orcover, (hemic,,1 inacti""lioll of LKR I may interf"r~ with ils physiolo{Akal 'lIllagollism of .,jgnals from growth f.U'lnrs. insulin. and onmj\('I1es. Tht." CL)~SK nk!.:h·! ofturnnr snppH'ssors ,)Ii r('f(H;.siv(' gt'I)(,~ ~tlr'Hlk1h.\':' llhtt bi~llldk: m~H.:ti\"J.tion i~ Ht .. "CSS~\fy fur ttmlOriguH.'~i~, (i-A, Th1~ Hh.Jde! flh Rhl, adtnil!11atnu;.; fh~lypi)~I'" (oli . .:md I':l,? in ::nany f:~;nlh~d ~}nd!'>p(lLl{Hcc.ulfl'rs (1. "0, P,lL1doxic.lHy. tllmor~()ften rd,lin fHH> ttWC thm~\l aile-It< or ~ornL' tumur suppn.'~sur gCIH.'~. (~g. j::Klpl (:)), plwi-phJ­U;<;. c kn"in homolog ~/d, l.;'.;:J~J C",1-0. and (·\'I'nt.t.'"d {9J. SW h h;~phHn~Hf t~t.:it'n(~' d(.·\'i~He' fnllll Knlld..,nll·~ nHKtd (10 -12); su;gt!stinp. that t~k\t· p~HtH,ubr ttHHil! .;,uppn.'\\'uP. m,l~ .:.:ut:-tumh t(). !n-..Ktl\~\ttl)n !Hl·,U ...... "i,.', th.lt <11'(" t'tislind from gClldlC or ('pi?,t'lwtic l~\ ... inn;>;, {L1). Rc\\·nd:., V.,T ~~b.((}\l.'~cd lh,lt biDlo~ic~;t!} ll'k'\~111!< (lKlllk~!lly rl.'''H.li\~ .. lipd~ ilLl(U­\ Jkd l1w p:i; Ph)ldll. \t.llh tllIHlinn:i1 \,pn'I.··'1U1.'nn'." t'{Pli\,lknt 10 Hw toss ot HilI.' ,dHdt..' 01 !ih' p,::; ~ ~1.{:I1C f H. L,)). '] 11.1t pbscisation, illong \\'ith lite ll'pmtuJ t'xu.'plinn~ 10 l\nud .... ll.11\ hypolht'~b c:,~) ,md dw fH('CI.'· ~ n'll,> '.'Juri-; Wd\ "tJpporh'd In PM; t}'f' LJm1t'd "'bh'\ r\:bht Hf'd~rh -';'i'"tY'H'\ Gr.mh H(! 1 Ailb! W itnd POI' AI NIT} do!i th~> tfunl .. m~n (,",nct" I tillndittu)!l 1tw >:q\hnf p(~h IkJliol< of this <)rf~de- wert d~h.1i€'d ~n I),);ft by th~ p.)ymtnt of PJ9'~ (fHrgC'<;.. H"I'> Mfld(- m~Jst thCh:·1orP. bf.' hf.·f(.·br mil!i<.0d'" advNt'{emi'Of h, ')(('old.").f'.;\-~'" w~!h is USC. St-( tinn ) 7 H \nl.t"ly to indh <tIt< {hiS f.<1t 1 , Suppof't&d by J predoooral ff!How~h;p (mIn the Ar:ll!t!<::an Ft!t.i0datiQ;"; f01 Pha!m.;;:::l'"~)· th::~! ldu<atioi''L . Hok'kth".z.D(-(:GI{'!\llilndklaW.Sm;t!1ChiJit tift CIlK(~1 RI;,.'""i;!drl,.k T owllom (O'lnpo~I(J'~'II~.f \Imuki tw. 'H!dfjf~~KJ' j):,,~ Of'rln ,m~J Iii", VI}. "imlth (h"ir uf (l'f'I('~~ jiYWt:lI< h, Hung~n.<n tan<tI"11nsM.1.J1t~ . .JO(IO Clf(,~e of Hop~, Ut1lvt:r ..... 't'i f)( l.}t.,h, ~d!t L.:tkc (~~ty, lJ I <3411 i" ~,S;'O. lv.LSOl 581 620<U'<.'IA. BOl·SaS OOll.lmiij:, hOl·lk,fi:;q::<illn-:.kl0/;d.l.ltllh.t,:du. 2598 ':;':." (knt of. liprd"'lhH ti\'<~ting IKB kit'!!n:-:.e (J6., 17). din:'ckd our .ltkntion to LKHI. " 'l'<mtlr di","t',ul tUllil" SUp!',t<,,,,,, ",,,odall .. l with ['t'ull' k!,iw .. < Inm;,r!'H1L> '; Ildmm~ 118.19). LI'HI h,\ nowl ""'dn"it!u-("mnl' kifl..~Sl' :STKl nat tlH' ;\pex l)i;, signaling (,;u;cadt.' thin '(~nM.·~ {<t'llnbr tllt'l ~~ IH.Hl)i.:n\.,t~l<~ ,.HId ad1lt~t\ an~lbo1it' ;,wd i,.<th.lOoiit.: p'OU .. 'V'>l'\ (Fig., 1), r ,}\Bt. an A\11) .. kiIU~i' ~in"\M' .:~nd tltnHII"Stlpprf'ssor. is ~1 Hl1iqut' link 1><'1"'<'<'11 "",Iab,,)k <111<1 pmlili .. ,\u'()nipol . .lfi.y sig.naHn!! (20····24). We "'I""llh,lf '\''''';\t< lipid 'p"fi," ,O\;,)unly flH\dili' LKBI ,,[ " Hu,k,,­phili\. Cy.\:·" FI.·,iduc in its Jdi\·,)thmloop. then~hr inhibiting. hOlh tht' pho~ph{H"y!:tti(ll~ 1)1' :\ ,\1 PKn .. ~IHd iht.~ th)Wfbtl\~;{m pro[laga!ioH of ~ig· ,ulsthnHlgh rh .. ·If<-H1-:\Ml'K,,-T'>C1f/-mn )R-Si,K fJ.!;\··.,dc.l)iSntp· tk.n 01 ~lIldhoh~ .md t .ildboHl 1Hl[lh::O~lC1~b i.mJ the f«ilur'l" to rt.·~lr~lill irUpPltlPI iak P'{~h.<ill tl-.lII:l..lJtt\lil <':OI.lJJ t.ontfjbllh,~ to hil1n~1[tmll .. l inl" m;~t1on <wa the 1kl~hh'n('d rat1({'r l'bk iii p\·utz·j(·ghrrs $yndrnli"w. Ch\.'illlt .. ,i i{hu,ti\~Hi(ln {lr"tullior ~uppn ... ~~tH prott'in::.-... Hkt· LKUl ~md p;)J. (Ollld be <H1 t'tlohlgjl~\l f,.H.tt)t' !" t.h~pIJsij .",nd hy~x·rphJ~l.j <.bsc)d.lko ,\'ith n\"1..>rl·xpl't.·.~h)J\ I ~f (:t)X 2 or cnl"onk int1i:unrnation that l\ln (>XP~)~C (1,:11 ... til t"4.,:Jdi'.'c !ipid '{l1!1 k.,. (".!-'), 26). EXPERIMENTAL PROCEDURES :\tH~ nld'~·~Snppb .. ~ ust:~.1 w~:n.: Duih"'('(.I'\ moddk<l Ll~k's lHl,dium ,\Ild '.;iJPplf'nwnf.:..1in','itr\)~;,'n); novtnt" in~ultn J,nd ftt">ntJmkin (111\'1£]"1) ~(IIi: PC, '(.'''",,\1\ ('h~l1m"b!: ·l· I I'.:!. al1d +ONI: ((Jym;", Chcmi- 1:,,1,,): -,-,nl~Wdi ml;L'/('dt,,,,~ -l .,)rht n::~HlHdt.: nbof)udt..'u!")'idr i ;\ICA f~) rroron!tl Rcs-s:;tn'h (JH.'mh·~lb 1m'): Compldt' \~ protease inhibitor IIH)lHtl' ~md FuGE\lF (~tf~m~felU{)1I r~ag~llt fRodlt' Applied xit'w:e); po!~\'h~n,lI ,Hlt ihtiCht:" din:t'tJ.:d .1g;1l!\~t 1.h:Hl. pho);,phu- Thrl '::" (\.\(PK(), .-\\[1'1'," I'hospho-'wr"·:\Cc. ACe. phospho.Thr"".ShK, S61'. ERr.:. f'i;;, ,\I'T If<-Kn, IKI'Ydnd COX,'2 ied! Slgn,llillf! T~(hllnln~ll"l; '''1'". mynn {(;t,H S~~!lldlini'~ '[·t.t.hno.;ng'''-':<i); h-lH'!"-\'r,M,li:-ih lwn'xida"c'u.~niu l~.Vl'd ":{,'<'lIIf(LIf~ ~HHib~lk, fS;11I1~~ CILU giott'dlllolo~y); !'\'l>l: Oll'iH' hr<Hw'" M\d \\-~·:..i~·!'n Lightin}'$,; \~ rlwnlilumi!wsf,~n«' H'Jg('n1S : t\-rkinFhm" Lift. S( H:IH. t'!'); IH.'l1lr.l\'aHn ·con.iug:oI.lh.:'d b{:Hd~ {l}il:HVj; !h'l! ·~'~H.:h.Hl!ntl ('p!l~,!>~' ·t,lg{;t'd t lIfi\H Ut1:--. 11)[" LKfi I (~df~ flom 01 ldnJl ~L1k{'I,~, l 'n['d>r~!!~' or ~ kl· .. n1kL FinLmd); ,1/1(1 ,1 Qt1ikCt).lt1~w· 'd ~Uf' dir •. :dl.,d llIUl~t~\.'IK",i~. k~t i\udtagl'J!<,.,L ("111 ("u/!rjrr'-- \fCF·' hh\bt (,lIh t'!' rvlhU\TCCI \H'l\' n1.linr,tint,d ill minim..! l',-"sl'mi.d n1,'(Jium J! .),7 'C in ,) hurnidif\('d innlt"hltor wHh "')'\ C(}., i h~ nh:dj~l!n \\ a~ ,:--uprdl..-'1l1.t·nt-l"d ,,:tth 2 tn,\! L ·~h..lt'Hnin\:, 1.:) ~.'Ht\..-'l :'JIfJm111 hi( ,nhdn.\tc, 0.1 t11\l nnlw~'.rnti;d ~lmlllO .H'lds, 1 nl\1 :-.ndmm h!" ..thbltfviJt;Wh I''''''.;! iUf'"' AMPK'f, AMr·.-J(fj';l~1~d kiH'J\.<:"~f; A((, M~tfl· .. C()A (;i'llg»(" yl;)~; MCAR )·<)mll1,):m~d~loj";<·<1·(MboKJ:lnldl) HbOntld~~lde' APB, arnidopen-ryl biotm,·1 HNt ·1 hldn.:1)l.y Z r'iOnC:nat IKK, hd3ldfl3H,':; NhO, nt.!CI<>llr t~l(tor JoB. -1 ONE.. 4·oxo'} nUtlt.":et P(; PI<)$~d91-djHlil1; 561<.56 Minas\:, h. ~hrortlIN~dr~; rnTOR, JIIdlrl r!1.-1h"m t.ugpt d r,~'MH1ydn: ERK, Ht~~{f:'lbj.H \~gr\.r.lI,u"9UId:I~d kin,a\t>: JNK {.jlm NH .. t>:'rn)lf\JI k~rM~~; PVOr, p~.lyv~')ybdenc d,flwwd~·: fL~, (CI,l! {..}if ':-ffvm: HA.. h('Ji);lqqh .. ltu',in.;P 'nl!m,,~opl((lpitdtiofl:, NA, nevW}vi-di("l, 48 Cyclopentenone PG and 4-HNE Inactivate LK8l/STKl1 Growlh factors, Insulin ~====~~==~==~------- Energy signaling -~. AMP>ATP ~ Acetyl CoA Carboxylase ----- ~J'J ~:;..,. S·I::;I~ oncogene SmTORI t:ATP Anabolic Processes Catabolic Processes --I KATP nC;UR[ 1.lK81 lign.illing pathway.lKBl ('STKl i \fUl)({: 1f)~.H JJn AMP·ki.sHo;.t!- kindl'!.(" in u~II~.Ani\'ityo' lKBl i\.lilllitt!{J In ~dl:;,with f)d~q~ilk AT? Au.umul,Hionof AMP,m dddititm of 11\ rlHmr-lH A!CAfl, JOI'.'<Hr!\ ttw A\1P~k;na~,,~ J.mMw iKtiwIy of U(BL whkh fpnwm, AMP'K(t .... " pho~pho.rhr'u AMPK", In rum, prh')~pho'Tn:t\") AMPK;l rmWf'rt\ A(C .-. pho~pht»!;,'r .", (l((:t{'(oA {Jrbl)~yk'lst PhOSDMo-rhr~ fJ. AMPK~{ al~o pho~DhoI)1at~s T){1!2. whKn johib,tsmTOR·n"lI?diatedtorwerSfon of S6K • pho!.pho- Thr"Wf j.6K. c..€'Us ~.!!,n${> (hangc'f, in !h';::-:f Ai\·\P 'AIP (JtIC M-,d ust" ~hc LKB~ ."., M.'H'Ku kInase (asc.ad~ to I'namtJin propet .lnabolic.,...,. tM,)oohc homeo5t"1"'~$. LKBl h~lS."!I dual role- as a tumor 5uppn:'SSOf Jnd (!'Wt;l!10,,":: (f<IJllb~ra hy ;u',t;lIJ'·If'l.Jinl) ,hr' ph0<;prMtidylifl:!);,>itol 1- kin,l5<t:'·t\Kr p.lthway, 'Nhkh prOp;lg,)t(>5 C)fl:abl~"~c ""tjf'li.'lh f{(lm ilv:.ulin ~1f'ld pm:lk'I,'.JW"Jrl <9nil!-; fwtn !)'EWl~h (,.tr.!urvrjf>(f)"iJ('t>c., P~IU\,Hl', (Un m~ Illl h~l\iflt'ln:-'li!in,()Jll rng'ml gent,)midn, ,lnd HY\; (I.:ul bf.l\l!1t.: "i,..'rum, 1~r..U ltl!oll c.~nC(f (.t.:. Hs \"'\'1\: lt1.:..wHaint'd in rninunal ~,:~~~t'mul medium ~Upph·llH.·nt{:d with L:; :~ .. htl't sodium bic;lrbonatt" ~}.l :11,\1 n/Jrh.' .... ·yt';Hh~; ,unin1) ;1t id:.> 1 if lH\l ~odium p)'fuVa(~, OJH mg/mt l1l'nnnl(l!1. Jnd :0", kt;d ~Y1\'nh.: \('rum. In u'rL.'tllll)Xpenmcnts. cdh w\:.,'rt' irH.:uh.lkJ 111 ~t'rum depk,tl'd nH.'dium containing 1% ktal bovine "'~'ntJn fill" (, h pnn!" lo !r~:'lPT';t:nh (k,,)-l"nhn:l ht.'low. ["dat;on ~)( l\:n:'1v.(' ("ul'ldtm/r f.m\di'tl ')), PC;· !\mifiop1'mylbiotin·· .\lC!··· ,md [U .. U td\ \~~'11,.' tff,!tt'd with IO-,f)O /.t\1 PG1\lA~Hnkioptm­t~ .. lh~lH!n \ It' ...11',), I)C! :, .. m11..!npt.'ot:'I1:Ht)!in ft!r ,t h 'l'lw (db \"',,~J't~ tyf-,(~d III .~,")1l m': !'!.HTt),l.{·. ,")0 m\' Tris. pH -,-L .:-) rn:,! \{gC! >. 1 m\! [(lTA, 1)( CtHlll'!de I q prnl'l;\"lt.' WlllbiloL .!. 01.\1 sodium lltHHlt.h:, <HH.I 2 n1.\( \ndium nrthn'.',ln,h1Jk Tlh' k .... Hl'''" ""d'" son}('(lkd H)~< tor t So ",t 4·' C. .A\fkr ('l'Hlt i!ug~!! i( In ,It; d,(N)O ~'i< j'll 11) milL ~~~mples. ((HH.lini[~g 1 00 p..g of Plotf!H) !'liHI! II fU: i..t.. E !,~ ~.ll ... ·"" \l,l'tI,.' 1t~-(Ub~ll!..'d \vnh 100 ~d of !1(,uu<-~~ vldm hCOld:-. in I ml ~l! phnsph,lh'·hl.llkn·<I ~ahw\\'ith OA'\) T'n\'l'n 20 for 1h h .11-+ 'C. Tilt: ..... \i;1i.lk' .. \'·;t'l!..· dh'n c"('!'!uifugt'd alSO'lJ >.. ,~ for~) rnin to hoh,tl'lH'lHrJ\'~d:n h'nfn, ("nrnpit':\~· ... ~:",:,-\ pulkkJ\"t1). Thl! h(:~~d~ '• . ..-eft \\",hiwd thrn'i:nw:--: \~ith 1 ml o! 1'lUbplul.t' bLdkrl.'d.·1.]lint'.n.4(S~Twe!~n :?U j!1l' ..... IHlp!1·.., \'d'lC (li.."phdl in ;·)H III p( L:wnltn!i In~H.Hng buffel ,OS',~, {3- I1kn',lptp('rh,'<no; ,ll",d h,','th'd:1I {r~'(' f(lj' hi 111111, r~r()teili !;{l:mph:"" (Fi fJ.-t:t \~I . :·r': f:adlPII.lh'd h .... SDS P:\.Cl. ,md tltlllsfl'Hn.llo P\'DF mt'Hl hr;."i!w~. '\kmb[.w~><... \\~'Il' hl<){ tnt With S'\, \\iv nonf/l~ dry milk in Tlt~ T, dwn inl Ub,Hc(! ~(tr 12 h ,'it i (: with prim,)J'Y ,1l1tihn(j!es dirtOct{'d ,\~,lliN LUH \l.lOIXl)'IH,,, i: :Hh)Oi, IKK y{l:j(~X)L I>iK (l:lOO())' ERK II: :0(10;, ,m<1 .-\~, [' II~)\I'. t"l1m,·,'d Iw hurs.'r,Hlish p..,."Xld,lSt'·nl!1ltl· r CBRUARY 3, 2006' . .' ~: j.e...; c;1;"z ~• galt'd, g.oal .1Illl·rai)[)il '"WOld,lt)" 3rtlibod, 11AO(~1). Antigt'Ol Jntihl',h wmpk-xe; were ddcdcd With \\'c$lern Li~lllin,,:" Eel. rC,l';Cl1b. li'(m.ljediOiI LKl$l IIA or l.KIlI (CllUSi 1-1:\ was t!,,1tlsicct<,d mto RKO cdl, lIsing I 1"~'I"Il.l:-:A, ., .d Lip"I;'(\<lmilldO(~I'-\1 j()r ·20 h folioll'lI1g th,' l)1,\!\UfKttliW', protocol. COX· 2 was lr,\I\i;«<ue'd Hltu Mer 7 (dis usin~ I S-<W s-<I D~A, :, iii l.ipof<ctarnindOilO i" for n h j()llowing tlw mJnuf.:H.:tuftr':s. pnlt<)(o\, Transfi,·(t~lln t'{fi(il'll(Y \ .. 'J~ IHNs1lr('d b)' im!llul1"dwmk,ll ddl't'min,l!i<1l1 of COX 21',"01";:1 III ("l,11 lysntt<s. Sampiils wen.' lysi..~d and fr;lct!O!Mll'd, ;.is ~\b(}\'I,,', and mcmbr':'HH:s \\'t:rc inn,lb;,ted for 12 h ,H AJ .( With pnrn,u>~' ~~nhhlldil'\ dlrn:h'd ~~~aln"'! C()X·2 (LIOO!)). S!tf~dltt'(ttd ,\iU([l,Wtri.. ')i~-:\ C210S L[,Bl muLlnt "'\.1~ (tlP~trut'luj n:-:.mg ;1 QmkChGngc~ \1 ~ih··dir'l·( ted rnut<-lgl..'nc5.'s bt following th, rHi.Hlubctul"t'I"'S proh)(. oJ. Residul' 2jO \t;,lS-U1[lrt'!"lt'd lrol11 ~l (.'ys hl.!)l'r lw u TCiC ttl J TCe substitl,lIpn. Th,' Id"lItlt:, "ftlw PWliUlt 'c,,,' <"It· firn:wd hy D>-:A ..... t'qlh'Hcing. A.\IPI\{t, ACe, find S()l\ P/,u.'>pb.JJ)-/(ilioJl A.~'d:;·.\ h) \\ '{'.\!CHi Hin!-­Mer' ··"'""' (eil:;.; \Vt..'n..· H',:,lh'd ,\ul1 o···(~) JtV "j the df~lt~.n;)~~·d P(; ..... T~, ·i H;"":r. or ,1 (H"';F Jo1'·.{ h unh-;..,:~ mlwnris~' sinh-d, rol;u\\"in~~ thi:-. innt h,HiwL'} !ll\t AI( 'AR rr~}. dll :\\·IP ll)lrnl.·til, \\;\', . iddn.11D cell:.., !~)! 30 n1in. In (~'rr~1il1 t'xpl'f'iH1l'mS cdls \'0.'\'1"\'" trl':Hcd with hl) II \[ P(,. tnr,~ 1~, and:;O 11\'; Wp.HH}Ul1 ftll" 2 h prinj to A1CAR tE\.':.nlHl'nl. Tlw cd I!:. \\1..'[\' ly~\'(J ,1~ {tL.'~(rjbt:d elhoyc Ll J(L! r~f P(OkH) \\.h tr:J(t.ionated hy :SIb" PAC';L, and rm)tdH:l '.\"('rc n,m~klTcd to P\'])F nwmbr,H1i.'~. Tlh' nh'lll l:n,lnc, wn~ Plllbnl 'Iilh P' illw, ~ antib"ui", Ji',l<;kd api",t pht"I'I,,) Thr'''·;\,\Wk" (1.10IM)), tnt;,] ;\\11'[.;,,· (1.ltXk)1. ph'''plw·Sd· '·;\U. 2599 49 Cydopentenone PG and 4-HNE inactivate LKB1/STK11 A. ERK2 HUMAN ERKl HUMAN ,TNK :4 HUMAN JNKl HOM.AN M3SB HUMAN HKll HOMAN !lOtA HOMAN LUll HUMAN AIlTl HUMAN IIIKG HUMAN CQ·n$(tns;t.lG B. we ~IA Pulldown FIGURE :t. CovoilJltntmodifiutior'! of (.eUul.)t kin.as.e':> by PGA l'ifmidopentylbiotini (urnp.aris.on of LKB 1 with otn~( ~!I!dnli::lthr~Qnine kirh)Sl/s., A );Q\",I':; th( {m'limJ,.Idd )";qU'--,'Il': INK, t t\K plU"AKI, !KK, ,~n1~ K!H kfn2l\~<;. {)'~' KHl U)(»~ "'rrl'~rm;"'l~ H\how~ tr'lPlw'l"hl0t<; dwt,l:it HI\, 20 p'i PG/l" lYle h;r~t? 21) jt!) PG:\ J!Ylij'Jfxnt),lbI0:in Of 20 ;;.'.' ,"(Flrn~,f> t(} ~f'q:>~>(,t~( kjn,~<,t>~ 'Nith i) h'(;rjf'l i'11;tOPf' i'';(((':-tli.:i',i ,11' 'Y"'i~ tmfYI th~k nvuuvi·dill <JlP!?.:):>(- bt.'ad~ INJ\ lful IK{ -·"d I ',8) (:~lf:.tJil!('d d ;, ;,r ammOp-pf1 I I::! liifJ), ph/)'-,pb ',. 'j hr "-p:'O St,}.: {1: :,O'OU)c ,md Wl~tl ;)- \I ",hl<- (1' loon;, il_, f''.. 11t~'lblutin undt'r these c:tp;.:rill:ct::,d '-- nllJltidll~, \\' ,--' f'lhui !led ~jrn- {plin'.u_',j bi.' 11,11 ll",U\t:::; for .112 .. r)G) , ,IIYJlciopc!1!\ lhi',\!m {li.1Ll P(lt i·1O ';, ~\lH.1 thi..' lh.t h .. md t() ,J pi..'tt.l·nt~~gc 01 thl' nmtrol. LxpvnmctH:; \\\.T<.. :-.. r'"Hci :1, [() tirn,~'", L'«hl dilLl depict ttw mjds-('Dt; d gl; tb:a,fun:, l\(.' tLnl;--;,r'i~'(,l",,'d dl<..'J1l V.-ttll FL·.., thc' ~ ',HT'.':.ponding. C'!lOS mut~1nt. PG,\ .ilm duprul;, Ih;, Ifill Jnl rl)!:,1 p!·{I.'~t~-lgl(u!{lin L_, and /)_ } 1_1,"IUiI:,'UJi Ln/i ut"di'Jr'ltll'd t{l<t:-.:',~'-';'" ]>(.;r .. Hl~II~(-II) IllHllUno~~~;s.lY~ \\i'iTC bUl th}t t1'o,--' LJ·.l)l d\.,]!' .hlduct with tsiki l~'pt;' LKHi Pl0;(-;1l :1'IUt;Uk( prot('lll (Fig. ,jAL Adduct k,mhl )(100'1''\\ ior :N h (li.lfOW;J!~ \ !I\ 1\\HlLbi II,H~:'f":- l't'otoct)L Cdb \'I:~;--rt' n' ••. ';lkd \-\'!r.h 10 jJ.\~ CO\ 2 ;;ihJbH •.) ;' >-~~ },:j\~ r(,l\'t;),Hl Chemical) or \ l'ltid,' t nnl rol f~-Ir ! ') t'oll( \', <-'J : Po' ! It"i! !!WJ 11 \\ I~ i\ 11 \~ \,)(;d,[,;'Khidonit ,l~ iJ 11.. T'(~r'~ UA ka dnd PC)j)} \10\ 1'::\:,1'.;:'.> L\'~ ~J~.I'd hl!!u'\\-;ng th: IH,\JllJl',H ,,~di~lrl' W-,h ~,,)mpkd ;md <l!v!ly/t'd ,I: ;11 finn \\1111 . kB1 \'.-i"_ Ojl <.'d('pi( nVf'f\».pl~·~:-.iof', II!- !IJ~ LkBl p\'lih'lrL »(1,\, ~1:niij\)lh'nt\:lhjotm t'orml-'d ':lJdud.s \vith \\dd !I,-P"' I.KHl ",\;,,,,.,, d ,d., i" \ICF<" ,",II, iFig. ,W:. Addi""".tll:" ~1(idud tnJrnJt10)) l .... n,_,t lU1l<,jU,-' ?G-t\ ,bbc,th PGA~ ;m1Id~)lh'IH~~)b; olin ,H'I:I ...l!'_:-P{~j, Ii) dtin fOr'lnf',i :lllducH wilh I.kHI in LlIw". ;,)ld j ~":-'u'>~ d~-pil ~ ,I (Id '. : I HilH·j h~ ItILI\;.!1 i! Ii',! 11\,' i l'llu!.11 ; WJS ,1~):;C:,s'._'J hy dll,:h ',I', 1"< '(,l.rJ,.in' ( A:\lPKn, BJs,d I~'\-d:, p[ po:-.1>fllH- tl'-,: ',,: (!llllI),I!l":"I" RESULTS the' /\,\11' 1,( .. -'; t(l/ {) h c!0PI\.'l'ltl.'!lt,llll,.' FC; .. md ---1 I i>~f n.>,_~(·tl\'t.' !q_~J\j ,-i. ~md 6) S;;;W'l;-!l dilfl'fi'H( t·ln:trr..'phftF: {J ( .. \rhon~_" tnc]l',(~ln~ ",\;,1-P(i! .J.!~~-P(lI;,.iJnd 1'(;,\,,!I~hih­d( ir..-c' ofU<31 in \KT<7 (lAb ·,timu ,:k,j ;1)iti\~11 W:J~ (()nn.:ntr~lIiOlhkpt_'n(kJII iFif~ !ht·">-l'lipidl., 111;ty lI~tl-dif~ lo\l~~'r :-..i'tilli. \\ilh ]'11 n:-Ii dnn ·"l-H\Y, tu the C~·~., n .. ,~>jch.jt.' in Jl<k IYLJ ,) '_lIW:_[tk Sfl'lne thl't./-i)n~nt.' kin~ls(.' (umor \UPpH';... ... ur, !I.I:--' ,{ 11'.1, t lUI ,tli!:u,", \\.:ilh I h r~sjJu(; III trll!.' ~\dl\'~Hil,'-,n \"''-<'' ,; Il;j-..n :u-,;i in (\)ntrlA~t. [Ri-.:. I~Jk. Pl)"iit!()Il (rig, 2A), Tn dct\.·tr~::·I'>- '.,;h\,t1VT (],_,_'tl', ThillI.' PGs {J.HI,-d t'f..\lct diredly \dth LKB! ,Inti othd [1(;,\ ,tmid"}lfCTnylbi,nrin -\ pnl PC: .,uuli\~~ h.l~, th' dklr:.t\,'IYri~,t!_l: ' h r n.n PC;,-\ Apr~ ",'.'111 :--'Uh~,Ci __ rJCnt!' q lit· !)~ . \"> 1<1 I :ll.'ut','I' ;dit I h·. \ld~, ther!.'l!} (,fLlL P(';.\ \i,' ,11, and 2 . llU\l;nJllnhlb!uun, l1kiHl -+. SI,) of 2b.,s -~ If. l~~ }I?II .J.l:'· )(;!!~u ¥.).3.0 lA/J..\!~lvH;..:t 'J J.!I f.L', .j i.l\T The dt'!"tl'(lphilic JJ ''-<l~'bun h<,:'c.ru:\c U1)H'm:rin: lipkb, XII.. lLid : ", [,I" rxlL 11!lt' ~uld IdtTl.l.ltl(dllOll I I ~ :l(:,\ ,It;il,I\~lW~l1\'j!w,)ln; FSj(!~'d PHX<;.ItIl-_d ~nld di,.,L<I, (~,y-,d('litly hlt.h \ dlul,u ;)d :l<l<n \!>.T, <,' Jl>:ky (rii, . .lj{ II,'" jl..,l , .... ,th I]~Kl:L 1XK~, " i:;, h iil,' di~tjnl"li ... ·~> C\-... H:'SHLk (urrr.::spllnd:ns hi m 1h..I<n (,'y-..;' In lJ~B!. I hWl:i.,~n-'r. tlwsl' kjjlJ';,,~"'" do h;\\,-'i' (\" ;,,"~;,;,Il'" -it oth~'l p~,~:\i1:on:;. 1'hl.l··..,. (f,1\Ui:J1 dl,1 11'_i-; n_\H.1. inj;y:"IlmllMtt.-i 2600 frol'n A\ U'r<n. Fu: plltl~pht)J:.Llljp'l ,,·1 \('( \'...-h (.'>1"1.,<1 <; thi~ {I bl;1,,' 1'".'\ j.) 2 I'(,i .. :md ,j J [<[ tnhd"t",\lh" ,\ ,ldh11;lk f(H ph(l,.,pi~j)-TIH-·;-,lY;\'\;PI'iL ~• . '\ 1. Likt'wt...e, si~naling through 111,' <\,d IH".l11dd pf phospho-j hi ·>oi) . rEBRU,lRY 3. 2006 50 Cyclopentenone PG and 4-HNE Inactivate LKB 1/STKl1 A ih ~ a; ~O :eIi I..tJ 3O~l- '" '" Ol 1 <L .. <f ~ :t<i <i.e « a:lC a'a' ~2 NA puHduwn lK81 IP.HA lKB1 11(,liHl Covalent modifkation of lKBl involves i-b (ys_lt!;) re!idu-f!'. Ii HKO 1 ,lnd 2i. HA t~lgg'(:d lKB 1 3 '-1,~d Only tYPi~ I !<;!'l 1 ~Aut>n!, lKBl ~vjth ,1' (2105 )l;b)lituliVl Jid not fUflH cll', JdJt,d I,.\A irwnuH,,)blo{ uf 'NikJ 1Yf,.{1: I Kfl! f.~( I -J (',',ll" r(;'.tr;~,i tt:r ,II):H 17 -,( with 6.-fl It 'f ('\t j.-'{,A~ .."lL2 pcn·\!,'( '')'.l.i) PGj)·!\PfHWC10Iil."4). ':;ih,t:.-' (t>-III'r'~_':ltt"; ht'iHh In i!'.n!.HfI f.1m' lUlh lont,limm.J <llHuttn eJ.H~q)t'.liliW\)rL,i;.h.:l'> "11:),,.' ~fhJI !uflm.'J ~lIl.H.ldutt wllh P(lA,·APn f:\!A p!i!lduN", ;'{uU;')' .~t d '\ 1 :·h'lj)·A"I~ 1;,,<\ fAllld(;'{"n.- i(itl;:> ·1} 1 ,I .... \~ j •. 11 thc ,'pl:'_~: 1_11 tilt-' i\ \, j 111< n iurnL.ltlun t,f ,u;.-J.,{_)I!I.,,·JI:. ,J"lJ~\ rhl':--'( :W!rlC ,:iII I',,\"!'I in ,\ 1': PC1'" I] lunl Td !flO;; di.,.!,ai I -J Z;{d~" li."ln~i ... tt..~nt with 1-:1'()"({01 H'ygfHtl~t'~ 2- in iJ1ll1lWHalil)n and in A. 8. c. 2 3 4 AMPKal .. ·--.. pAMPKa I .!I!!d< PGAI AICAR '+ '+ 10% FCS 2060100 <3 20 '+ + 5 6 --I ~r_ I '+ 1% FCS C PGAI 4HNE 40NE F'~ 'IRE 4. lip-ids with oElIE<trophili< f~-(dfbQm lohibit :''''fineithreonin~ kin"~fe ",{ttv· ity of ci!-lIular lK31. A, ~,/( ~ '/ ff'lI '; \\ i it ':ck-d ·;.'<h,dhl't nl' Jlot l,p~ji. Ih!'''.t' "tng~dH' M tl'·;'~'ld~ll.rl ~WP!',l iil') lI';dd!Vii~'m '1if'~'h;m-~~m Ifh.'\)!\'lin ,\1 ~-",'li'l LKBl H 11' it" d! I ,i\l-~l. Ud'~I dl, lidi\, lli',I',III'" I: ~ )111' :'\ . l,ltl( In, \\',J."" \'h.'L;(~~'Uh'd II}!.!"" ,tt'lL ~l knov:n lIlhihlh'>! '<',jth ..:'. rn\' >\iCAl.: (hf,. ~\ \ IJ,iil ,;llrcil'; '.dh{ ()\")~i.Ild\lq,>'P[\.'nH:'fdpd \,..,.ith ())\-) ::1.1.:1.' :d:.ilJ(;U. !]J:;1l ~HI}d'-'-!J.-IJh!i·(t,d f"lhk'j,: transtt'''' __ ~lAd ~-1CT - wn('tl !nt.'dXth:..l \\ Jf lL'hldl.l!lic ,Kid IT:1bk, t L \)S ,3\iK. ,i "1,,,;,-,'11:. pho:..plh}--! ht Cn\<~ it~hhi:nl', ',d ti(~r . .Ilhi P(l!.), 1~1I'rrLltion Ix:, u,n" ild!)'~j, i It,d ;[flRUAR{ j 2601 51 Cyclopentenone PG and 4-HNE Inactivate LKB 1/STK11 I A, , I, pAce I ~ "t.~ 4Q "!~ , I I ,~~ , ~ I I !I, 4 "'BV~'~~' I S6K I psal< 1-*-- c Phospho-p70S6K I 150 I !:~:II.t_ I I I i AICAR . • I I I 12-P(lJ, Rapamycin • . L I l(,UfH: ~J. Ele-ctrophilk lipid~ inhibit phoiphorylilltion at acetyl.(oA carboxyhu,@ and 56 kina •• downstr.am from th.l, Inhibtllon 01 lKBl .. ,. AMP kina •• _ A Immu­nobl~ 1r of tot.li A(e ,l'l<.! phQ"ph.-'I <;N~': /1,(( in Iy~arf.'''' from MfF j (;:ih; inf.uh,lf.-.d w;~h hi) w.t PGB!< P(;A 1, -ll ?·r't;J~;.. 4,H.'\(, <)1 M~';S0 to 10'\·) ",'~hi( 1(> I (J!!!rt11 tIJ' ·1 h "'ld tht>n ,'o'd"!: 2 !}W ./I,I(M~ fc;, 3fJ ni'0 t(l snf'!1uiat.:- the L';((P w ... /\\WK,t ~'9nJh~,g tiJ'lh'/(')y ~r'ld $ubs~qlJ('{'t /J,C( ph()spholybHol~_ R(>pn:'s('nt,}'ti\>~ ~ipids ttutr u"lhib!'tt.,':j tKB~, \.It tl)(:"~}~x''-( of {Itt· ~lylhr1iny \Mth-'h.l-)', dho ",lkn'.bJ:t:d fhl! pIHJ'1-plH.J!yld(itm of ACe ,~ ~tJb~(rcllt! 01 AMPK(I !ffg. 11.6. !mmunob!t)t oftOi<l1 S6K ilnd pho)pho-H1f ' . St,l\ m IY"'-ilH'~ of M(f.·/ -tell:. ;n(~lb"ted 4 h '>'"11th \'etw::l~ i~Gm:' 1f: 4 h '",,1h (('. iJ' ~ 11:P(dl ,~k:ne Um1!:,.'): 30 m;n wIth 2 IWJ. .~J(ItH Ji{lftc (101112 .:H. >4 h '."t!th j, i 2. PGJl .11)j then 30 minwi"tt) !'K,~R llan'.' ,1}. J. h 'will! 5tl Ij?) IdP,!It:yt m .~Iol\l~ U~,r.I" )}: u~ -4 II '<-'lith .l i l·rC)), p!~:" .' I! with S<J !\~,( fdlMmynn, plm .10 min wllh A1CAR lk'm{' f)J. (, hr.,tnqr<~!n of diit.111l fi lh!:' II~t~H"lly of !tJt> ~,6K<md phos.pho-lhr·"'·~)6K hand~ Wii~ q~N<,urf>d and dl'p~nf!od as d p~f((>nr3q€' ofth~ (ontwl {i (" 10t"h, ','orhir!f' ,llonf', iaM ii, Valw"', H'prt"s.ent m,p.·u:: v ).F,.!"i .1 t'\ple\'- {'( }X"l eH)' v.hij. 1'1 ,H ~ ilUlIh htJ pca·,' ,m-d P(;j): hI! .,.,~ JlI.h."i<'" h~ mo( h tr/m~tt'\ led {t..'II:o.. DISCUSSION R('·n .,. ntly, l.ABi W.h idl,.-'Iltith:d .\:-. till' ~I..'rk' n..'~p~)ns.ibl(' t()f P""Ul/. 1e:th. l".'lS :'} nd(oUh.. ' (].;--l, l~J), \\ hi'\. II pI \,:d i .... p 0\<.':-' to tUIl){)~:'" pt" th~. .' {L~\..':..ti'd'; tr.)cL rcprodudin,.' or~~.ln\. ,'Ind hrc.): .... r. Cant l'J'" lnn(knl'l" 111 i-'dHJ. .. lt:gh ers s~'ndrnrn~ i·; ;t···l~ idd e;n.'.1tl>r tihH1 in the gE.'neLll pOpUi.UlOll U'lJL ~\nJ it b tht.: on!:' tJnu!Ull..·,.H\{<.;r:<:.ynJroll\\,.' ~~ttrihuh.'d tp lp:-,...,.·pf fuw,..tkm mut.11l0nS m.J :"l~rllh' ·lhn..'OIllIK' km.l:'t', Thc ,,,,,'Bul.H' :.uhs;r,lh.'s ,md hIn l0t\lGil roks of LKn! ~I, ..... :n: unklhlwn until 10'l.)3· .. 1(}O·L \\h!.~n i!1y,,-'\1iS,j tlWS dl~{"q\'('rl'd th .. ,t 11 phu<:,pl)(~rY{H('d A\tPK ~:n1d tnndllHwd (lS ,1 uniqtH.' A.\IP 1.i!M~t' killJ\e l.20--2 .. U. LKR] kt~ tllln \ ll~l] ~t.'lluLH roiL'~ .t~ tIlUO\\~: Hllll\l~ "uPP!l':-'~,or :..w d fcgubto}' ~)t <Hl.)hnh~- :(',~r:tl'h)IH' ik)mi'fl~;n~ls. LI\H I ,.whw\'t:,,,,, lh PtI.rpos!' by nl'g,~ti\"d~ rt.·[.!ul,1tlo1:!- tht: f'hl~,ph..~tid~Jinositoj 3·kiu.J\C'-:\['T p;.nh '~"'<J~ I.FIB- J J \\'hl.'!1 .. ,d!Vdl.l'd h~ thL'u I !:'fll'Ul\ C "liHWb, LKH! ,11 ~d t\I'T ... {'nil opp~)~ing .-..igtu1.-.. r:"l,;t ddE.crmine the Lttt() of dw :lct!V\' in,ldh'I' (Hnn:-.nr·JSCl·2, .Ilt"lOH, ,Old Jiho-..nm;JI :-'hK. tilh-. 1lI hll~:, h.\bml'~ ,l!ub'ih' ,wd (.~L1hnhc PlYl(1 :..':'C~ In onit-f ru nUln',~'n t't·!/;d.·H l·t1l:q~" honWd~,Lt .... is {ATP il'\'d:-,J (..l)). Our tbu ::-.how tlFH ~::.:ogf:IWH"" ,A ... ,·ctro· phiLt.. hplll ... , ,J' \\dl ,p. -i.:lldngcrl!!U" (d<d~:-.h h} (dlul':H' C(),\·',~, {,HI inhihit Lt.:1H xdidp,' ,1nd :-;hift rhl' t·qudihrilHn il11iw p,Hln\',lY lIn",~ld ph'y:,phnr:'htioll ,md ~lui\.lt!I:Ir; pj" ~ibo~\)nl.il S6K.1f Lhi~ ~+nuld O( eu!" \\,']):;-11 ~ i'lL, kt\t.· hal !!ttl.· ,\"1 P t (! '\uppnd pHJP~·t hdl1, .. l.ltiutl of [(\:\ info prott'in;;, it misht LH i!i1.lt~' t'I.JIHIH· P/(tt?,l·",."jl,H1. i )~· . ."r{"gubti'~!i 01 pruh . .'in l!~Hl ..... bltjl!1 i~ IIUIHI11,lnt tTl ';"\"t. l~d <-,{lln.:!:. (31)-3.1,) ! .• w. ~"\i~!l'''''l(m ()t J.!<BI u1 tn(':1st 1\ln1nr:;, l:'i .~'~SU(l,:1!('d '-<l!h puu]" prYJ!!.l1o::;'J:-' ,Hid ~Ur\~\',l! cn} <llh..t \\'dll til\..' i.I.Hl··dti'l~l 110m pfl' I1uliglhtllt tn Ilulisn.mt ~ttI!l(J:' 2602 A B AICAR 4 5 100 + AI'. • • • IIU)I1(;' Inhlbftlon 01 ,.lIula, lK81 ~ctlvity by ""topl,.lIy "p,."ed <y<loOXY9e­o, se-2. A hi'Swqr::'lrn dtpk:t\nq Ihe relative amount of pho$pho· Thr~JJ AMP'Kn in MO-7 ({.jlo;, t!.;l{lsf(>(h:'d with.1 ~n,xk fCHlstHi(t (.; fn (OX) \ ! and tH'·;Hcd wilh {f..') fbujJfOtf'rl (fL.-tlj ),1- tJ()~If~ ,·dkr h"In.f~>( lion. (!;-;'1k ""'*..'W ~r)\" I.Jh"'!'N~ f~)~ 30 IBin wilh mj-.'d~Utll u.mt<lln In9 1 0',· t(~ ...... ,~~h H}O F'l (~: Ihuprofen or "'i.'hir If' (J)nlu)! fnHo'l'lf"d hy., h of l[lo:.ubal!On 'ovltl1 !l'tO jo('.' ..ij,)i.nidvnl{ .. H.~<..lI./lA) 2 HI',' AI(.,fJ.H ..... '.:is .;ld{k~d to.:lH cdh to '.itlHwI4h':- U<Bl ,!drYJI.,. (db eAW\.'~~iH.1 lU, .... · ... .J1Il1 !rl.:dlt'd \:\iitIJ 100 ,v.' i.II.:d)iJunk <lud Shlh'';1.:d d dp(f~~hi:' in t j{,B I ,H.ti .... lty d~ lHp.ct')tHt'd by ptHHpilu,AMFK,j lOn!~nl rddlil>'t' lu ';.onlro!. te!is: er.pr~m.ln9 C01·2 J;"d.exposi;!'d \1:) 1 Ot) pJ..' rP)'lbuprof\'n ~h.)Wed J sigorflc.:mt r+Xov· <::ry ,r) lKS 1 'Il.;:~ivity J:i {tH,~J:So;;.H('d by phospho AMPK~f (O!l1p;.w~d with (elis, r;ot treated '.\'IIh jtn,pHlr~·n. 8. ~q)H'~~>:n!dth,~' ilrlf;~tlflObl{)l ~how!r1<J inhibition d rlw phmphorylt1· wmnf AMf'Kil bl';(ij\II;\~t)f (OX,l tL;I/l'if~~(1lnll .'IndilrMhui{Wll{ ~nd lW;nrnf'111 dod wnw· NY of ~he phosphnryl,\tion of AMPK(, bNauSe of preifl(uh,JtiOI'1 with lbllpn:.lif.'rl rh~ 1))~<'I"l<;Al oflhf' A:\WK,1 ':H1d pho<;.pho lhr' '0' AMPKfl bands was m~asufc-d .1nd d(>picted ,) .... ,) !)\~!(f'llt~~p.' ()f1f~~', /)!If'n!~),' 1n n·:" tue/l,R ;Ibn. ... , hJJ~ •., n V;)lw:''; H~f)H'>'>~)~~t rf)f>.jfj , S.r., /l t TABLE 1 Prostaglandin formation by MCF·7 cells \',I:~:"'" il"~i1,"n' <..;<. :;""l':i:rjtl~,.'~d ... \1Cl' -(.I!·,~\.t·!I·!I.n)'.l,'!.~.·d~~j!l,!,t c( } '\ ), 1] ,~ n I'" ;, '.! !:~'·tl t· •. { .~ l h. ll~::- ,.lih'r t, ,m .... k1 (i'ID. ~ 1.'1b. \\U~' in~ l..lh~~<,.'d !~~r 1 II ",dt td:,I.H' illti..JW), l.""!,>!Jh.:" n'h~k lJl!O ::-\\ .N~. edt:'> ~~t'lt' lb.'lll.~, ... t b,Hn1 ,dtL [on W .• r;,rdiid'J!I!:" :It.id ':\;\ll>:'~ In!:!. ,\;\(.1 k\d:-Ilt PCi ,wd P{;P in thl' mn.llU'1"I '.'.1 r ',' ,.: .. !l::llil J b'< :mln,in~U:--"'.I~·, \'dw:k llJ<;ljJ,\!AA l\.lO p \1 :\A plu .... :\~ NS -~.------- \'t+ii I~' :~;:~:::: :~:.~ ~)lu~ ~~. YJM 9,'}" :U ;.0 - N,.! J th.-:" :. . 1.. L3 \Im:k 1.5.7: L) (,I').': .~. rH,l ;·j.<J1 ) ~U 18.'; < 9,2 'M,:'," Wl:,' ~HL~ :::: ,i(,~ COX v 2 Cnm!olt"(:tiull 1':;. Hd i(,:' :. 2.·t ~\)2:,' .:. 121:;..6 iH .. ; .:.1:1.1..$ !.~r~~\\"th in hmg ('.ll~{·t·r (.:f; L ~ Ill,t lnkr{',ti ngiy, ~t'\'t'!',l! !'Iudit's shov,,' th.if h.Utl.tlI.UI!'~L'" (P.!..', t.·Xp1t' ........ (J )\·2 j {).. 3{1), ~Uld ih t.'.\lH\'~..,ioll Lt!.."ihutl..:"'," 1UHHll"!g{'nl·",j";' \'L~,~ h.W·l,in o \J).:"l··;Hk llOJl 1.·1 ,., ;If\ Hlol)U :0Xjl.wlh i.' (.\7, .~X). Alth()u~h .... pt!t. ~!J.Hi\·t.' ~II tl1:\ p·;)ilH, our n.>!-"ults !-Ut!.gt.'\.l :lb.lt inhihition oi !.hF.llJl,.'! <UV Ilf !l ... d~l:r!l!(,d in,1~fj\',ifllll) h~ It.·a,fh:f.' hpld "'Pt>(ll'''. 01 \n·\·l"t<l,t"Jrc~':-.tlm col Cux :~, ... lh h ,l', 111 Fi!',. h, flU?' hl"1\\' t"nnSntw:1H ~,,~ :-"lmil~ll t(~ h\::.~ '.)1 <.:X;Ht".,:>\!t\1l 01 Lk.Bl. or IIHJtaliuu;.11 i!1J.c.:tl\"~ltion, 1 h. i'i t 1'.., a tnmn! ·.. . llrJPf(':-.'~·m 1"hi~1 (ltkn rd~t1f)" f>lH" wild I} pl' ,t!!I'!l', d",,·,.i,~\ing hom l'mrd~·pn', ... >"'U hil hypodw.,.~~. This ~usg>l.'~h th~~t LKJ{1 i:. ,1 ~)<}ll,'lltl~d (;indid,lk ((\I in,I:I.."tjv,\tillg prou::,.~i..'~ lhat .:.ire di~tinft hom gClW1"l( lit" cplgC!h'tii. k·~il"H1 ..... rurtlkITn~H'~" \\"f' r~"'l:'()lkJ tlut f .kB I w'iuk! hr...' \..~ .. I\ .. .tklltl: IiHh.lifil..'I..l <.md irtJdh'dtL'J by (,ydoptHtL'BPtH.< Plt 52 Cyc/opentenone PG and 4-HNE Inactivate LKB 1/STK11 dth .... f~:.;< hn v)."~tj)pi~',, ttwy inh:!u lhl< hUI not 1,\'[" ~ l6. 3,_n, 1';'1.'-.(:..] llit :"iu..:-rlin .. " I,,-,J mut~;t!',·lle",r~. ·,'\,)('rilHcnts "'\'Jth dl{'(t and fl\l( tl\\' \ !\_U\J,nnlt!". ;:)dlt.'{" uLtt b'hi::-, f,))' inUhitic\J) n( !}._Kn ,11'1'.\ H-J .. ,t> fkcJ.lls(~ LKfH is.l r.H\' t':\Jrnpk' of ,)tUrn!)!r "Ilppr{'\snf, -whh"h functions .15 ;.\ scdne/t hr('~)nlnl" ki.ru~;,;.-' ::; K. L))' 'X.,:' \.\ )m~);,IH,-'d tht.: -t1rnino add <"CqHdK"-' ()f Ll<H! !hnk~n~: [nop '<'.lth thosl,.~ {t)f IKK, ERK, ]\'1\, /\KT. ~Hld pJN, ThIS ~,cqunl( (' ,-)h~~ntlH'll1 IT\,Cilkd ~l Cy!O..Di.' residue in LKBl th~H UHH::--POIKkd t(\ l\:s~dul...' in lKK~ These Cys ft.'Sl(h.!I.'S- .IT,; d;·;tmd to LklL. iJ"':ktl -ltd !hJ"':j.> hut not F.RK, JNK, ~md p.3-K kin'l~>\':'>. wfudl hJI,'f.' ,\ ki ,-11 l.n~ In corrqnr,)bh: p{)sitwn~ (Hg, 2). Uur rr,;'':>ull.s ~h..:.r\\ thal ,'\,_l"T,uttulon<..' l'!~~~ ,)f l:K A- <:.Hld ,i'S{Ties Ct)\\ih:tul\' !Hndlh 1 KB! .lId 11<1'11 hill nl)~ rt~~l ~)) '\1\>\2, IKKy :\1'"1'; Cy" h' rq;'["H cd b} Stf~W, Thl1~. i'l'!!uL\J do ~[(\t a\td !ndl:-o(dminakh \<;:tl' 1)(;-\ .}\nl~ q,'!'t<'xlkrimcnttd Lunditll'l:h_ 'fhl' r"'.lil!-, i~1 III ttlr 111110'.\'[0;!.' tlit' ph<uJ!l~,\..u"()~j\..J,1 the 'j,nU\\-Tl ~ lH'!l)H ;JI n',h'L\";l'\ h, .'11 '1ll>,,;.llULHt,d (;trbollyls (\'lid-1:l~_"l t\<,({li(lll'i- 1]ld ~ ;-WtiI!O clC;J~ lin S.U'l'l<: ;,!(u.. '''>:-..Ihfiil: pI' Ilw rlH d il--. tfw-,\ (I~>~H '-~"';!h i \';;It;,dh ,di lipId", b,;)c] t1l \ "lJubr ptntdns by t\'\\:l '~Jbk, 1 J :ikr.;mt"l), For ,'i-ornt: lij>I-;'\: .. \\illt ;tll (L/) llli:-dl \\'11h pr~)I~ ,'.,' IrK'II", 1,llI}. Cy< C:\'-," in thin). pf, ,\l~J -;k(ll. l.]~(\\t'><...-, .qidlUI, I.\L1: ~ __ k; tl tlun E)Y'h' 1H.)fl-ir, (I-t. ]-, I,) oll'll'n.:'_\j\lt'.,,-iidldC()\ l'k( ~(ni'dllh "p;_'( )~ __ " COil r.n~ : k,cj\)1h: wi:l \'{'Etl"lh~I;\. ,d'finit.) inklutlion \ rl1phlhc J~' radxHl neat ,1 p6.'> ~ubunH ofNFKB , III ':lth('PStn (42), and lipid itdJuct Thu:-.. th!uh, ~tI'l.H,~_;lr in!!w.'n(e, "md lkk !ii)I, \ ,\1'1 H'<Kt with more -,;r~>ss, rnfLlnHll(Jtton. rn,!}' coul~'iin muhipil> \\ ah ;:H1lr,I3-.-tHl.si..\tu~ ".-dly, I) .my p~)tlwphy:;:io1oil, :U',,-' jh"~'n dd:c'l tl",-! Ph'd( Ii,_" <11 SikS cd int-brnm,) tJO;, !,lr, }-•. )), [n,;{..";,ti~,,\til;-' I pUll:V<lVd that the,,.: rt~mJiVl: lipid :-.pni,-·" lh. 1p 1i' ..... oLl· nIL: ,!,11)d,l 1 (l!'..Ik!dJ~, modifying l,d~ , i-';,'g ': i\), ,)P), '~lkh . \,d,NiW,-!~·d:~lIlt'I;{ H '\> {/;,uJ i; j'fI((ldlli_t /.AUI. r[CRU/diY 1 2006'. ,l:; 11'." t .'_' [ 1 [ 11'~:- 2603 53 Cyclopentenone PG and 4-HNE Inactivate LKB11STK11 ·l2 t :t'.1bb. L \\" ,{) .\d, ). Mir;\~~~, \L \\"'n.r. [C >.md Kdtf. H. f (hPli Fmtem .\n 11, ~:q --X-in H. 'hhat~ .. T, Y.11)\.,id"""l,, ;·,h.;. L K;.::nl',tlJ.'lX:a. ,\,. ;-':",tJlni.:.:14. H" ,\'!,lY..ltJH", H., Y~).,;k~i, L .. mid Lfbti,~, K. \2~))H!!. litH!. Llmn. VS. 2·fu}.~-2f;:·H:Y~ H L,(\'~;n'.:l\: ,A, L LJnJ""L :\., !~J.rrLl~.·h:::'fI~.h~n . .\.,(",.j"",:"f. L k. P~~.kr;.;;.:m, rJ. A, I.m<.>nl, C. ;\\,>'ft>~\.. : ~ L ,,;:J :>,,~; .... ," t..·~1~"'1, \' M. ';;.\~! P H(I.>:;h':ih r 373. '1"7-i. J~; -1:> :\!d',:il<;!lt A. L, Lfu ... tnl~H, \. .< Lld')~T. {}, ( .. ~md Hill.lJ. \\ :,L,jll'l! 8il"ibm~'·1.r~ 4:.t. ·kNS:···l,"lfi.S 2604 .'fft. (1~H1. L ,\1,)nllw.' ]1.. <\l1d Hnh·r::-. L.. L !l <>fr'r! ri hi tltrtn l,{'.l. Hk'ifd .. ·H!;';t">~ "$,'. tJ~~'I'. \"., hd':Nt. \\ L :~;Jb::T'. L L. : . J~(i .\h~n'~\~·.: {) j 1 wt- ffY·.:i"!H~. B{'1lj~?}·, A"fal4..16, S-:x)" 53(~ .;~, ~~Hbi'lt-J> 'i '. Kn;)l.h!.:-"L (,!qw.~" 'i., ;(,lHi~,ILt< ~,. :K~jh;l\,<~"+~' \1.,. l-i:!d l.\hd~-i, i'--., ~ln,I()! l.l\i<~!, OkltJ 177.1114>'1· liJ,~'A~ :;:~, b;-;:,~t\l'k", H, ~:~ U. }1,. ,);,d i.,l;tl',,; ,\ ",: 'i'>~r :,.r~a<a ~(.:-" IJ8, ~2,-) .. ,;~3 :,v. t J\vr .. ·IH~·. L \V:b:q.:h;:~', P .• \ .. ,~ml G:lI'>Y, D, W. \2i~X~! X;~t... {\>~';'. i;f<!NlUi<~ 2, :~;:~" 9'; .. ' rEBRUARY 3, 2006 CHAPTER 3 REACTIVE OXYGEN SPECIES FROM CYCLOOXYGENASE-2 AND 5- LIPOXYGENASE INACTIVATE PTEN TUMOR SUPPRESSOR Tracy M. Covey, Kornelia Edes, Frank A. Fitzpatrick Published as: TM Covey, K Edes, FA Fitzpatrick (2007) Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor Oncogene advance online publication, 19 March 2007. Reprinted with permission 55 Docogene (2007), 1· 9 t,. 2007 Nature Publishing GroliP Ail rights reserved 0950·9232107 530,00 'ltww.nature.comiOl1c ORIGINAL ARTICLE Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor TM Covey', K Edes2 and FA Fitzpatrick' I J)el'drlmelll (!( Medicilllli Chemistry, Cllirersily 0/ Ulah. Salt Lake City, IT, lSil (///1/:1111/11.1111(/11 C(/I/('cr In.witufe, Ullirersily (!i' Utah, Silit Lake Cit)'. liT USA Cyclooxygenast'-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes are owrexpressed during inflammation and multistage tumor progression in many neoplastic disorders including lung, breast and pancreatic cnncers. Here we report that the tumor suppressor phosphatnse and tensin homolog (P'rEN) is oxidized and inactivated during urachidonic acid (AA) metnbolism in puocreutic CllDCcr cell lines expressing COX-2 or 5-LOX. Oxidation or PTE~ decreases its phosphatase activity, favoring increased phosphatidylinositol 3.4.5-triphosphate production, actinl­tion of Akt and phosphorylation of downstream Akt targets including GSK-3/J and S6K. TheSt' effects are recapitulated with pancreatic phospholipase A2• which hydro­lyses the release of membnlDl'-bound AA. Interfercnl'C with PTEN's physiological antagonism of signals from growth ractors, in~\Ilin and oncogenes may conler risk for hyper­trophic or nl'oplastic diseaSl'S associated with chronic inrtammation or unwarranted oxidatiH~ mctabolism of essential fatty acids. Ollcogcne' advance online publication. 19 March 2007; doi: 10.I03R"sj.onc.121 0391 Keywords: Akt: PTEN: cydooxygenaSL'-2; 5-lipoxygenase; tumor suppressor Introduction Akt is a serine-threonine kinase involn:d in metabolism. proliferation. motility and survival (Fresno Yarn ('/ a/.. 20(4). Regulatillll of Akt requires tht~ participatioll of several dilTcrent proteins. Following stimulation by growth factors or other mediators. phosphoinositide ,1-kinase (pI3K) gencmtes a lipid product called phos­phatidylinositol .<.4.5-triphnsphate (pIPd (Cantky and NeeL 1999), Gent:ration of PIpJ recruits Akt to th..: plasma membrane through a pleckslrill hl'll1ology (PH) dOll1ain. At tl1<: memhrani,!. Akt is fully acli\'ated <ICter (\IfTCsrond~nc~: Dr F.'\ I'illp.llnek, Departmenl of l\kdicin,!I Chl'mi'lln and Om .. ,)lt)}!il:al Sci(·I1~\.'s. thlllhm;m Canl'l'r I n!:l.1 it lIh.'. Lni\'~rsil;' of Llah, 200iJ Cirdc of Hope, Sail l.ake Cil~. LT ~41 12, LSA. E·mail: frank.til/pulrkk.iI h,'l.u,,,ll.cdu Rl:l'~i\cd 16 '\\)Vcmhcr ~O()6: 1'1:' i~'d 25 ,hH1u~lry ~()07: ,hxcptl.:d I h.'hruilr~ 21X)7 phosphorylation by phosphoinosilide-depcndent kinase­I (PDK I) and PDK2 at residLles ThrJ08 and Ser473, respectively (Chan cl al .. 1999), Subsequently. Akt c:an phosphorylate maIlY downstream protcins and thereby positively and negatively regulate diverse signaling pathways. Akt signaling is restrained by PTEN (phosphatase and tcnsill homolog ddded on chromosome 10). a phos­phatase and tUlllor suppressor that dcphosporylates PIP, to I'll'" (Lcsli.: and Downes. 20(2). By reducing the levels of PIP,. PTEN inhihits the recruitment of Akt to the plasma membrane and prevents its activation. pTEN function is regulated by post-translational modification. cellular localization and redox modulation at the active-site cysteinf ,( Lee 1'1 al., 2002; Y u ('I al.. 2n05; (iericke e[ al .. 20(6), Arachidonic acid (AA) is an essential fatty acid involved in the in!lammatory response. AA is metabo­lized by cyclllllxygcnase (COX) and lipoxygenase (LOX) enzymes into prostaglandins (PG) and Icukotrienes (I;n. respCi:lin.:ly, Tht:rc is increasing support (0 show that essential '('tty acids stimulate cell growth: however. the mechanism is not fully understood (Marks ('I (// .• 2000). Recent reports indi~'ate that AA caust:s an increase in phosphorylation or Akt. although the means for this is ullclear. Some reports indicate that the activation of Akt is PI3K dependent (Hii ('/ III.. 2001; Hughes-Fulford ('{ ai.. 2006: Wildroudt and Freeman. 20(6) and others report it is PI3K indcpendent (Gorin ('( 1/1 .. 200 I), SOll1e reports indicate that AA mctabolism is lH.:cessarv (Hii <'I (/1 .. 200 I: Li tInd Malik. 2005: Hughcs-Fuirord ('I III.. 20()6) and others n:port that AA m..:tabolism is not a factor (Gorin c/ al .. 200l). MOllnting evidence suggests that both 5-LOX and COX-2 illlllli.:nce the progrl'ssion of several human cancers (Aggarwal ('I <II .. 20(6). including pancreatic cancer (Dillg <'I al .. 2001)' Using B\PC3. AsPC I and PL-45 pancreatic cancer cells. which have COX-2 and;nr 5-LOX exprc;sion. we n:pllrt that AA metabolism leads to :1n inefease in Akt phosphorylation. We show that the I1wchanisi11 responsible for (his is oxidation and jna(;tivati(lll 01' PTE~ tumor suppressor. Oxidatillil of PTEN by AA ml'wbolislll dt:creases its activity. ckvates 1'11'\ levels and incn:ases signaling through Akt and its downstream targets, These effecls an: r.:capilulatcd with pancreatic: phospholipas..: A" (PLA2J. an enzyme thai catalyses the release of AA from llIl'mbranes in Oxidation of PTEN by AA metabolism 1M COI'ey et at pancreatic ducls, Disruption of PTEN and other tumor suppressor, by labile mediators may contribute to tbe etiology III cancers ass()(iated with dmmic inflamma­tion or oYl'rc'<r'rcssion of COX and LOX (WClgner ('I a/., :0(6). Results Pali('/'(,lIlic ClIllcer cells cunp!'!'.,s COX-2 alld 5-LOX CII::)'IJICS Ahnormal 'exprCSSi(lll or hoth COX ,lIld LOX has been reported in pancrcatic' c~lnccr (Xiong. :0(4), To study the CrfeCh 01' AA meta holism on cell signaling, four pancrccilic C<lI1cn celllincs (BxPCI, AsPC I, Panc-I alill PL-451 were cbn,cn for their differing nprcssi(ln of (OX ~\Ild LOX Cl17:ymcs (Figure la), All ((1m cdllines csplT"l'd PTT:\, Thc Panc-l cells had no COX-~ dud Jittk COX-I PI :i-l.OX expression, thus scrvlI\g as ~I Cllntrol ,xii line with diminutive All. metabolism. The a R""> 9,,," ~<l" ~ <Q+ ~'" q~ qV PTEN --_. COX-l "'-..< - 5-LOX ..... - - COX-2 .. Tubulln •••• b BxPC3 cells PO. Ser·13 AKT-+ TotaIAKT" "... - 1I!I!I!i: -~ ~-. ASPC1 cells PO. Ser·?3 AKT-+ TolaIAKT-+ __ PL45 cells PO. Ser413 AKT-+ Pane-' cells PO. Ser"3 AKT-+ TotaIAKT'" .......... -­o 5 10 20 40 60 ~MAA 56 AsPCl cells have COX-l and 5-LOX, PI.-45 have 5-LOX and modest COX-I. HxPC3 cells have COX-l and 5-LOX with mode,t COX-I expn:ssion. COX-2 is the main source of PGs in these cells. For instance, BxPC3 cells treated)Omill with 60JlM All. secreled 14.4.t 1,9ng PGE~im1. IU ng PGD,!ml and O.8±O,Ol ng Icukotricnc B4 (LT13.)ml. These values wcre~5-fold above the cOlltrollcvds 2,9 ± .J.O ng PGE2!ml, 0.1 ug PGD2i mi and 0.16=0,04 ngL TB4/1111 sct:rctcd by Bxpe) cells in medium without AA (p < 0,05), By contrast, AsPC I cells. which express COX-I but not COX-2, secreted 3.0 ± 1.7 ngml PCi Ec and 2,{]::t 1.2 ng!ml (P > 0,(5) when treated