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Reactive Lipid Species from Cyclooxygenase-2 Inactivate Tumor Suppressor LKB1/STK11

2005; Elsevier BV; Volume: 281; Issue: 5 Linguagem: Inglês

10.1074/jbc.m509723200

ISSN

1083-351X

Autores

Tracy M. Wagner, James E. Mullally, F.A. Fitzpatrick,

Tópico(s)

Cancer, Lipids, and Metabolism

Resumo

LKB1, a unique serine/threonine kinase tumor suppressor, modulates anabolic and catabolic homeostasis, cell proliferation, and organ polarity. Chemically reactive lipids, e.g. cyclopentenone prostaglandins, formed a covalent adduct with LKB1 in MCF-7 and RKO cells. Site-directed mutagenesis implicated Cys210 in the LKB1 activation loop as the residue modified. Notably, ERK, JNK, and AKT serine/threonine kinases with leucine or methionine, instead of cysteine, in their activation loop did not form a covalent lipid adduct. 4-Hydroxy-2-nonenal, 4-oxo-2-nonenal, and cyclopentenone prostaglandin A and J, which all contain α,β-unsaturated carbonyls, inhibited the AMP-kinase kinase activity of cellular LKB1. In turn, this attenuated signals throughout the LKB1 → AMP kinase pathway and disrupted its restraint of ribosomal S6 kinases. The electrophilic β-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2α, and TxB2, did not inhibit LKB1 activity (p > 0.05). Ectopic expression of cyclooxygenase-2 and endogenous biosynthesis of eicosanoids also inhibited LKB1 activity in MCF-7 cells. Our results suggested a molecular mechanism whereby chronic inflammation or oxidative stress may confer risk for hypertrophic or neoplastic diseases. Moreover, chemical inactivation of LKB1 may interfere with its physiological antagonism of signals from growth factors, insulin, and oncogenes. LKB1, a unique serine/threonine kinase tumor suppressor, modulates anabolic and catabolic homeostasis, cell proliferation, and organ polarity. Chemically reactive lipids, e.g. cyclopentenone prostaglandins, formed a covalent adduct with LKB1 in MCF-7 and RKO cells. Site-directed mutagenesis implicated Cys210 in the LKB1 activation loop as the residue modified. Notably, ERK, JNK, and AKT serine/threonine kinases with leucine or methionine, instead of cysteine, in their activation loop did not form a covalent lipid adduct. 4-Hydroxy-2-nonenal, 4-oxo-2-nonenal, and cyclopentenone prostaglandin A and J, which all contain α,β-unsaturated carbonyls, inhibited the AMP-kinase kinase activity of cellular LKB1. In turn, this attenuated signals throughout the LKB1 → AMP kinase pathway and disrupted its restraint of ribosomal S6 kinases. The electrophilic β-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2α, and TxB2, did not inhibit LKB1 activity (p > 0.05). Ectopic expression of cyclooxygenase-2 and endogenous biosynthesis of eicosanoids also inhibited LKB1 activity in MCF-7 cells. Our results suggested a molecular mechanism whereby chronic inflammation or oxidative stress may confer risk for hypertrophic or neoplastic diseases. Moreover, chemical inactivation of LKB1 may interfere with its physiological antagonism of signals from growth factors, insulin, and oncogenes. The classic model of tumor suppressors as recessive genes stipulates that biallelic inactivation is necessary for tumorigenesis (1Knudson A.G. Am. J. Med. Genet. 2002; 111: 96-102Crossref PubMed Scopus (147) Google Scholar, 2Hansen M.F. Cavenee W.K. Cell. 1988; 53: 173-174Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 3Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 820-823Crossref PubMed Scopus (5519) Google Scholar). This model fits Rb1, adenomatous polyposis coli, and p53 in many familial and sporadic cancers (1Knudson A.G. Am. J. Med. Genet. 2002; 111: 96-102Crossref PubMed Scopus (147) Google Scholar, 4Vogelstein B. Kinzler K.W. Nat. Med. 2004; 10: 789-799Crossref PubMed Scopus (3302) Google Scholar). Paradoxically, tumors often retain one functional allele of some tumor suppressor genes, e.g. 27Kip1 (5Fero M.L. Randel E. Gurley K.E. Roberts J.M. Kemp C.J. Nature. 1998; 396: 177-180Crossref PubMed Scopus (681) Google Scholar), phosphatase tensin homolog (6Kwabi-Addo B. Giri D. Schmidt K. Podsypanina K. Parsons R. Greenberg N. Ittmann M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11563-11568Crossref PubMed Scopus (273) Google Scholar), LKB1 (7Avizienyte E. Loukola A. Roth S. Hemminki A. Tarkkanen M. Salovaara R. Arola J. Butzow R. Husgafvel-Pursiainen K. Kokkola A. Jarvinen H. Aaltonen L.A. Am. J. Pathol. 1999; 154: 677-681Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 8Esteller M. Avizienyte E. Corn P.G. Lothe R.A. Baylin S.B. Aaltonen L.A. Herman J.G. Oncogene. 2000; 19: 164-168Crossref PubMed Scopus (161) Google Scholar), and even p53 (9Venkatachalam S. Shi Y.P. Jones S.N. Vogel H. Bradley A. Pinkel D. Donehower L.A. EMBO J. 1988; 17: 4657-4667Crossref Scopus (373) Google Scholar). Such haploinsufficiency deviates from Knudson's model (10Santarosa M. Ashworth A. Biochim. Biophys. Acta. 2004; 1654: 105-122Crossref PubMed Scopus (165) Google Scholar, 11Paige A.J. Cell. Mol. Life Sci. 2003; 60: 2147-2163Crossref PubMed Scopus (43) Google Scholar, 12Cook W.D McCaw B.J. Oncogene. 2000; 19: 3434-3438Crossref PubMed Scopus (81) Google Scholar), suggesting that these particular tumor suppressors may succumb to inactivation processes that are distinct from genetic or epigenetic lesions (13Jones P.A. Laird P.W. Nat. Genet. 1999; 21: 163-167Crossref PubMed Scopus (2049) Google Scholar). Recently, we discovered that biologically relevant, chemically reactive lipids inactivated the p53 protein, with functional consequences equivalent to the loss of one allele of the p53 gene (14Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar, 15Moos P.J. Edes K. Cassidy P. Massuda E. Fitzpatrick F.A. J. Biol. Chem. 2003; 278: 745-750Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). That observation, along with the reported exceptions to Knudson's hypothesis (7Avizienyte E. Loukola A. Roth S. Hemminki A. Tarkkanen M. Salovaara R. Arola J. Butzow R. Husgafvel-Pursiainen K. Kokkola A. Jarvinen H. Aaltonen L.A. Am. J. Pathol. 1999; 154: 677-681Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 8Esteller M. Avizienyte E. Corn P.G. Lothe R.A. Baylin S.B. Aaltonen L.A. Herman J.G. Oncogene. 2000; 19: 164-168Crossref PubMed Scopus (161) Google Scholar) and the precedent of lipids inactivating IκB kinase (16Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google Scholar, 17Ji C. Kozak K.R. Marnett L.J. J. Biol. Chem. 2001; 276: 18223-18228Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), directed our attention to LKB1, a recently discovered tumor suppressor associated with Peutz-Jeghers hamartoma syndrome (18Jenne D.E. Reimann H. Nezu J. Friedel W. Loff S. Jeschke R. Muller O. Back W. Zimmer M. Nat. Genet. 1998; 18: 38-43Crossref PubMed Scopus (974) Google Scholar, 19Hemminki A. Markie D. Tomlinson I. Avizienyte E. Roth S. Loukola A. Bignell G. Warren W. Aminoff M. Hoglund P. Jarvinen H. Kristo P. Pelin K. Ridanpaa M. Salovaara R. Toro T. Bodmer W. Olschwang S. Olsen A.S. Stratton M.R. de la Chapelle A. Aaltonen L.A. Nature. 1998; 391: 184-187Crossref PubMed Scopus (1335) Google Scholar). LKB1 is a novel serine/threonine kinase (STK11) at the apex of a signaling cascade that senses cellular energy homeostasis and adjusts anabolic and catabolic processes (Fig. 1). LKB1, an AMP-kinase kinase and tumor suppressor, is a unique link between metabolic and proliferation/polarity signaling (20Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 21Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1418) Google Scholar, 22Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1047) Google Scholar, 23Hardie D.G. J. Cell Sci. 2004; 117: 5479-5487Crossref PubMed Scopus (957) Google Scholar, 24Kyriakis J.M. J. Biol. 2003. 2003; : 2/26Google Scholar). We report that reactive lipid species covalently modify LKB1 at a nucleophilic Cys210 residue in its activation loop, thereby inhibiting both the phosphorylation of AMPKα 3The abbreviations used are: AMPKα, AMP-activated kinase α; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; APB, amidopentyl biotin; 4-HNE, 4-hydroxy-2-nonenal; IKK, IκB kinase; NFκB, nuclear factor κB; 4-ONE, 4-oxo-2-nonenal; PG, prostaglandin; S6K, S6 kinase; Tx, thromboxane; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; PVDF, polyvinylidene difluoride; FCS, fetal calf serum; HA, hemagglutinin; IP, immunoprecipitation; NA, neutravidin. 3The abbreviations used are: AMPKα, AMP-activated kinase α; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; APB, amidopentyl biotin; 4-HNE, 4-hydroxy-2-nonenal; IKK, IκB kinase; NFκB, nuclear factor κB; 4-ONE, 4-oxo-2-nonenal; PG, prostaglandin; S6K, S6 kinase; Tx, thromboxane; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; PVDF, polyvinylidene difluoride; FCS, fetal calf serum; HA, hemagglutinin; IP, immunoprecipitation; NA, neutravidin. and the downstream propagation of signals through the LKB1-AMPKα-TSC1/2-mTOR-S6K cascade. Disruption of anabolic and catabolic homeostasis and the failure to restrain inappropriate protein translation could contribute to hamartoma formation and the heightened cancer risk in Peutz-Jeghers syndrome. Chemical inactivation of tumor suppressor proteins, like LKB1 and p53, could be an etiological factor in dysplasia and hyperplasia associated with overexpression of COX-2 or chronic inflammation that can expose cells to reactive lipid species (25Thun M.J. Henley S.J. Gansler T. Novartis Found. Symp. 2004; 256: 6-21Crossref PubMed Google Scholar, 26Fitzpatrick F.A. Int. Immunopharmacol. 2001; 1: 1651-1667Crossref PubMed Scopus (135) Google Scholar). Materials—Supplies used were Dulbecco's modified Eagle's medium and supplements (Invitrogen); bovine insulin and gentamicin (Invitrogen); PG (Cayman Chemicals); 4-HNE and 4-ONE (Cayman Chemicals); 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) (Toronto Research Chemicals Inc); Complete™ protease inhibitor mixture and FuGENE 6 transfection reagent (Roche Applied Science); polyclonal antibodies directed against LKB1, phospho-Thr172-AMPKα, AMPKα, phospho-Ser79-ACC, ACC, phospho-Thr389-S6K, S6K, ERK, JNK, AKT, IKKα, IKKγ and COX-2 (Cell Signaling Technologies); rapamycin (Cell Signaling Technologies); horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology); PVDF membranes and Western Lighting™ chemiluminescence reagents (PerkinElmer Life Sciences); neutravidin-conjugated beads (Pierce); hemagglutinin epitope-tagged constructs for LKB1 (gift from Dr. Tomi Makela, University of Helsinki, Finland); and a QuikChange™ site-directed mutagenesis kit (Stratagene). Cell Culture—MCF-7 breast cancer cells (ATCC) were maintained in minimal essential medium at 37 °C in a humidified incubator with 5% CO2. The medium was supplemented with 2 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 0.01 mg/ml bovine insulin, 0.01 mg/ml gentamicin, and 10% fetal bovine serum. RKO colon cancer cells were maintained in minimal essential medium supplemented with 1.5 g/liter sodium bicarbonate, 0.1 mm nonessential amino acids, 1.0 mm sodium pyruvate, 0.01 mg/ml gentamicin, and 10% fetal bovine serum. In certain experiments, cells were incubated in serum-depleted medium containing 1% fetal bovine serum for 6 h prior to treatments described below. Isolation of Kinases Covalently Labeled by PG-Amidopentylbiotin—MCF-7 and RKO cells were treated with 10–60 μm PGA1-amidopentylbiotin or Δ12-PGJ2-amidopentylbiotin for 4 h. The cells were lysed in 250 mm sucrose, 50 mm Tris, pH 7.4, 5 mm MgCl2, 1 mm EGTA, 1× Complete™ protease inhibitor, 2 mm sodium fluoride, and 2 mm sodium orthovanadate. The lysates were sonicated 10× for 1 s at 4 °C. After centrifugation at 10,000 × g for 10 min, samples containing 100 μg of protein from total cell lysates were incubated with 100 μl of neutravidin beads in 1 ml of phosphate-buffered saline with 0.4% Tween 20 for 16 h at 4 °C. The samples were then centrifuged at 500 × g for 5 min to isolate neutravidin-biotin complexes (NA pulldown). The beads were washed three times with 1 ml of phosphate-buffered saline, 0.4% Tween 20. The samples were dissolved in 50 μl of Laemmli loading buffer, 0.5% β-mercaptoethanol and heated at 95 °C for 10 min. Protein samples (15 μg) were fractionated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% w/v nonfat dry milk in TBS-T, then incubated for 12 h at 4 °C with primary antibodies directed against LKB1 (1:1000), IKKα (1:1000), IKKγ (1:1000), JNK (1:1000), ERK (1:1000), and AKT (1:1000), followed by horseradish peroxidase-conjugated, goat anti-rabbit secondary antibody (1:4000). Antigen-antibody complexes were detected with Western Lighting™ ECL reagents. Transfection—LKB1-HA or LKB1 (C210S)-HA was transfected into RKO cells using 1 μg/μl DNA, 3 μl Lipofectamine2000™ for 20 h following the manufacturer's protocol. COX-2 was transfected into MCF-7 cells using 1 μg/μl DNA, 3 μl Lipofectamine2000™ for 24 h following the manufacturer's protocol. Transfection efficiency was measured by immunochemical determination of COX-2 protein in cell lysates. Samples were lysed and fractionated, as above, and membranes were incubated for 12 h at 4 °C with primary antibodies directed against COX-2 (1:1000). Site-directed Mutagenesis—A C210S LKB1 mutant was constructed using a QuikChange™ site-directed mutagenesis kit following the manufacturer's protocol. Residue 210 was converted from a Cys to a Ser by a TGC to a TCC substitution. The identity of the product was confirmed by DNA sequencing. AMPKα, ACC, and S6K Phosphorylation Assays by Western Blot—MCF-7 cells were treated with 0–60 μm of the designated PGs, Tx, 4-HNE, or 4-ONE for 4 h unless otherwise stated. Following this incubation, 2 mm AICAR (27Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1020) Google Scholar), an AMP mimetic, was added to cells for 30 min. In certain experiments cells were treated with 60 μm PG for 4 h, and 50 nm rapamycin for 2 h prior to AICAR treatment. The cells were lysed as described above; 15 μg of protein was fractionated by SDS-PAGE, and proteins were transferred to PVDF membranes. The membranes were probed with primary antibodies directed against phospho-Thr172-AMPKα (1:1000), total AMPKα (1:1000), phospho-Ser79-ACC (1:1000), phospho-Thr389-p70 S6K (1:1000), and total p70 S6K (1:1000), followed by horseradish peroxidase-conjugated, goat anti-rabbit secondary antibody (1:5000). Protein bands were detected with Western Lighting™. The bands were analyzed using a Kodak Image Station 440™, and the net band intensity was converted to a percentage of the control. Experiments were repeated 3–10 times, and data depict the mean ± S.E. Prostaglandin E2 and D2 Formation—Enzyme immunoassays were performed to assess PGE2 and PGD2 metabolite formation. COX-2 was transfected into MCF-7 cells using 1 μg/μl DNA, 3 μl of Lipofectamine 2000™ for 24 h following the manufacturer's protocol. Cells were treated with 10 μm COX-2 inhibitor NS-398 (Cayman Chemical) or vehicle control for 1 h followed by treatment with 100 μm of arachidonic acid for 4 h. PGE2 Express EIA kit and PGD2-MOX Express kits (Cayman Chemical) were used following the manufacturer's protocol. Medium from cell culture was sampled and analyzed at 30 m. Experiments were repeated three times, and results depict the mean ± S.E. Statistics—Statistical significance was assessed by analysis of variance with Bonferroni's or Newman-Keul's post-hoc test for comparisons among groups. Covalent Modification of Cellular LKB1 by Electrophilic Lipids—Cyclopentenone PG and 4-HNE react covalently with IKK (16Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google Scholar, 17Ji C. Kozak K.R. Marnett L.J. J. Biol. Chem. 2001; 276: 18223-18228Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Thus, these lipids may modify other serine/threonine kinases with homology to the Cys179 residue in IKK. LKB1, a unique serine/threonine kinase tumor suppressor, has a nucleophilic Cys210 that aligns with the Cys179 residue in the activation loop of IKKα and IKKβ. In contrast, ERK, JNK, p38, and AKT kinases have Met or Leu residues in the corresponding position (Fig. 2A). To determine whether electrophilic PGs could react directly with LKB1 and other kinases, we used PGA1 amidopentylbiotin (PGA1-APB). This PG analog has the characteristic α,β-unsaturated ketone of PGA1 but a C-1 biotin amide instead of a C-1 carboxyl group. Proteins that react with PGA1-APB will subsequently contain a biotin epitope, which binds to neutravidin beads, thereby enabling their isolation and identification (15Moos P.J. Edes K. Cassidy P. Massuda E. Fitzpatrick F.A. J. Biol. Chem. 2003; 278: 745-750Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). PGA1-amidopentylbiotin reacted covalently with cellular LKB1 and IKKα but not with ERK1/2, JNK2, AKT, or IKKγ (Fig. 2B). These inert kinases lack the distinctive Cys residue corresponding to Cys179 in IKKα or Cys210 in LKB1. However, these kinases do have Cys residues at other positions. Thus, cellular serine/threonine kinases do not react indiscriminately with PGA1-amidopentylbiotin under these experimental conditions. We obtained similar results for Δ12-PGJ2-amidopentylbiotin (data not shown). Site-directed mutagenesis demonstrated that Cys210 in the activation loop of LKB1 is required for lipid adduct formation. RKO cells express negligible amounts of LKB1; therefore, we transfected them with plasmids encoding LKB1 or the corresponding C210S mutant. PGA1-amidopentylbiotin formed a covalent adduct with wild type LKB1 protein but not with the LKB1-C210S mutant protein (Fig. 3A). Adduct formation with LKB1 was not dependent on ectopic overexpression of the LKB1 protein. PGA1-amidopentylbiotin formed adducts with wild type LKB1 expressed physiologically in MCF-7 cells (Fig. 3B). Additionally, adduct formation is not unique to PGA1 as both PGA1-amidopentylbiotin and Δ12-PGJ2-amidopentylbiotin formed adducts with LKB1 in MCF7 cells (Fig. 3B). Inhibition of Cellular LKB1 Serine/Threonine Kinase Activity by Reactive Lipid Species—The AMP-kinase kinase activity of LKB1 can be determined by measuring the cellular conversion of AMPKα into phospho-Thr172-AMPKα. Basal levels of phospho-Thr172-AMPKα were low but detectable in MCF-7 cells grown in 10% FCS (Fig. 4A, lane 1). Phospho-Thr172-AMPKα levels rose in cells incubated with the AMP mimetic, AICAR (Fig. 4A, lane 3), and in cells placed in 1% FCS for 6 h (Fig. 4A, lane 5). PGA1 inhibited the formation of phospho-Thr172-AMPKα by these stimuli (Fig. 4A, lanes 2, 4, and 6). Several different reactive lipid species that have electrophilic β-carbons, including 4-HNE, 4-ONE, 15-deoxy-Δ12, Δ14-PGJ2, Δ12-PGJ2, and PGA1, inhibited the AMP-kinase kinase activity of LKB1 in MCF-7 cells stimulated with AICAR (Fig. 4B). Inhibition was concentration-dependent (Fig. 4C) with IC50 (half-maximal inhibition, mean ± S.E.) of 26.8 ± 4.3 μm PGA1 (n = 10), 12 ± 2.0 μm Δ12-PGJ2 (n = 8), 3.0 ± 1.4 μm 4-HNE (n = 4), and 2.5 ± 1.0 μm 4-ONE (n = 3). The electrophilic β-carbon appeared to be critical for inhibition, because unreactive lipids, including cyclopentenone PGB1, PGE2, PGF2α, 15-keto-PGF2α, and TxB2 at concentrations of 60–100 μm did not inhibit LKB1 activity (Fig. 4B). Inhibition of the LKB1-AMPKα-mTOR-S6 Kinase Signaling Pathway by Reactive Lipid Species—Inhibition of LKB1 by reactive lipids affected proximal and distal components in its signaling pathway downstream from AMPKα. For instance, PGA1, Δ12-PGJ2, and 4-HNE inhibited the phosphorylation of ACC, a substrate for phospho-Thr172-AMPKα, whereas PGB1 did not (Fig. 5A). Likewise, signaling through the S6 kinase pathway was affected. The basal level of phospho-Thr389-S6 kinase was readily detectable in MCF-7 cells, which must be grown in media containing 0.01 mg/ml insulin (Fig. 5, B and C, lane 1). Activation of the cellular LKB1-AMPKα pathway with 2 mm AICAR reduced the level of phospho-Thr389-S6K, the active form of S6K (Fig. 5, B and C, lane 3). Δ12-PGJ2, alone, enhanced S6K activity in MCF-7 cells (Fig. 5, B and C, lane 2 versus lane 1). Δ12-PGJ2 antagonized the effect of AICAR on cellular LKB1 activity and restored the content of phospho-Thr389-S6K to the control, tonic level (Fig. 5, B and C, lane 4 versus lane 3 and lane 1). Finally, rapamycin, which interacts directly with mTOR, overrode the inactivation of LKB1 by Δ12-PGJ2 and blocked phosphorylation of S6K (Fig. 5, B and C, lanes 5 and 6). Thus, by inactivating cellular LKB1 at the apex of the AMPKα pathway, Δ12-PGJ2 enhanced the distal formation of anabolically active phospho-Thr389-S6K, consistent with the scheme shown in Fig. 1. Inhibition of LKB1 Activity by Ectopic Expression of Cyclooxygenase-2—COX-2 expression and activity are increased in inflammation and in tumors (25Thun M.J. Henley S.J. Gansler T. Novartis Found. Symp. 2004; 256: 6-21Crossref PubMed Google Scholar, 26Fitzpatrick F.A. Int. Immunopharmacol. 2001; 1: 1651-1667Crossref PubMed Scopus (135) Google Scholar). This exposes cells to autocoid mediators and related, reactive lipid species. To extend our investigation, we tested whether or not transfection of MCF7 cells with a plasmid encoding COX-2 would inhibit LKB1 kinase activity by enabling biosynthesis of endogenous eicosanoids. Ectopic expression of COX-2 in the presence of its substrate, arachidonic acid, inhibited the kinase activity of LKB1. LKB1 activity, measured as phospho-Thr172-AMPKα formation, was designated 100% in mock-transfected MCF-7 cells stimulated with 2 mm AICAR (Fig. 6A). LKB1 activity remained comparable with control in MCF-7 cells transfected with COX-2 and stimulated with 2 mm AICAR. The phospho-Thr172-AMPKα content fell (p < 0.01) in MCF-7 cells transfected with COX-2 and supplemented with 100 μm arachidonic acid (Fig. 6B, lane 5). In mock-transfected MCF-7 cells incubated with 100 μm arachidonic acid, the phospho-Thr172-AMPKα levels were indistinguishable from the control (p > 0.05) (Fig. 6A). Ibuprofen, a known inhibitor of COX-2, restored the kinase activity of LKB1 demonstrated by phospho-Thr172-AMPKα content (Fig. 6B, lane 6). These data are consistent with the inhibition of LKB1 by endogenously generated reactive lipid species. We verified that MCF-7 cells transfected with COX-2 made more PGE2 and PGD2 than mock-transfected cells when incubated with 100 μm arachidonic acid (Table 1). NS-398, a specific COX-2 inhibitor, lowered PGE2 and PGD2 formation by cells transfected with COX-2 but not mock-transfected cells. MCF-7 cells constitutively express COX-1 (28Liu X.H. Rose D.P. Cancer Res. 1996; 56: 5125-5127PubMed Google Scholar), which accounts for PGE2 and PGD2 biosynthesis by mock-transfected cells.TABLE 1Prostaglandin formation by MCF-7 cellsTreatmentMockCOX-2 transfection PGE2pg/mlVehicle9.5 ± 2.118.7 ± 9.2100 μm AA173.0 ± 66.2368.7 ± 101.7100 μm AA plus NS-398136.7 ± 14.388.3 ± 36.8TreatmentMockCOX-2 transfection PGD2pg/mlVehicle13.7 ± 3.316.7 ± 2.4100 μm AA649.7 ± 139.1892.7 ± 128.6100 μm AA plus NS-398540.3 ± 21.1534.7 ± 153.8 Open table in a new tab Recently, LKB1 was identified as the gene responsible for Peutz-Jeghers syndrome (18Jenne D.E. Reimann H. Nezu J. Friedel W. Loff S. Jeschke R. Muller O. Back W. Zimmer M. Nat. Genet. 1998; 18: 38-43Crossref PubMed Scopus (974) Google Scholar, 19Hemminki A. Markie D. Tomlinson I. Avizienyte E. Roth S. Loukola A. Bignell G. Warren W. Aminoff M. Hoglund P. Jarvinen H. Kristo P. Pelin K. Ridanpaa M. Salovaara R. Toro T. Bodmer W. Olschwang S. Olsen A.S. Stratton M.R. de la Chapelle A. Aaltonen L.A. Nature. 1998; 391: 184-187Crossref PubMed Scopus (1335) Google Scholar), which predisposes to tumors of the digestive tract, reproductive organs, and breast. Cancer incidence in Peutz-Jeghers syndrome is 5–18-fold greater than in the general population (29Giardiello F.M. Welsh S.B. Hamilton S.R. Offerhaus G.J.A. Gittelsohn A.M. Booker S.V. Krush A.J. Yardley J.H. Luk G.D. N. Engl. J. Med. 1987; 316: 1511-1514Crossref PubMed Scopus (728) Google Scholar), and it is the only familial cancer syndrome attributed to loss-of-function mutations in a serine/threonine kinase. The cellular substrates and biological roles of LKB1 were unknown until 2003–2004, when investigators discovered that it phosphorylated AMPK and functioned as a unique AMP-kinase kinase (20Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. 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When activated by their respective stimuli, LKB1 and AKT send opposing signals that determine the ratio of the active/inactive forms of TSC1/2, mTOR, and ribosomal S6K. This, in turn, balances anabolic and catabolic processes in order to maintain cellular energy homeostasis (ATP levels) (23Hardie D.G. J. Cell Sci. 2004; 117: 5479-5487Crossref PubMed Scopus (957) Google Scholar). Our data show that exogenous electrophilic lipids, as well as endogenous catalysis by cellular COX-2, can inhibit LKB1 activity and shift the equilibrium in the pathway toward phosphorylation and activation of ribosomal S6K. If this should occur when cells have too little ATP to support proper translation of RNA into proteins, it might facilitate tumor progression. Dysregulation of protein translation is important in several cancers (30Watkins S.J. Norbury C.J. Br. J. Cancer. 2002; 86: 1023-1027Crossref PubMed Scopus (58) Google Scholar, 31Brugarolas J. Kaelin Jr., W.G. 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This suggests that LKB1 is a potential candidate for inactivating processes that are distinct from genetic or epigenetic lesions. Furthermore, we reasoned that LKB1 would be covalently modified and inactivated by cyclopentenone PG and 4-HNE as follows. PGA1 and 15-deoxy-PGJ2 inhibit some serine/threonine kinases but not others. For example, they inhibit IKK but not ERK1, ERK2, p38, or JNK (16Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1200) Google Scholar, 39Hortelano S. Castrillo A. Alvarez A.M. Bosca L. J. Immunol. 2000; 165: 6525-6531Crossref PubMed Scopus (113) Google Scholar). Based on site-directed mutagenesis and pharmacological experiments with inert and reactive eicosanoids, Rossi et al. (16Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. 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