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Originally published In Press as doi:10.1074/jbc.M509723200 on November 25, 2005

J. Biol. Chem., Vol. 281, Issue 5, 2598-2604, February 3, 2006
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Reactive Lipid Species from Cyclooxygenase-2 Inactivate Tumor Suppressor LKB1/STK11

CYCLOPENTENONE PROSTAGLANDINS AND 4-HYDROXY-2-NONENAL COVALENTLY MODIFY AND INHIBIT THE AMP-KINASE KINASE THAT MODULATES CELLULAR ENERGY HOMEOSTASIS AND PROTEIN TRANSLATION*

Tracy M. Wagner1, James E. Mullally, and F. A. Fitzpatrick2

From the Department of Medicinal Chemistry, University of Utah, Huntsman Cancer Institute, Salt Lake City, Utah 84112

Received for publication, September 2, 2005 , and in revised form, November 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha},beta-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 beta-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2{alpha}, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The classic model of tumor suppressors as recessive genes stipulates that biallelic inactivation is necessary for tumorigenesis (1–3). This model fits Rb1, adenomatous polyposis coli, and p53 in many familial and sporadic cancers (1, 4). Paradoxically, tumors often retain one functional allele of some tumor suppressor genes, e.g. 27Kip1 (5), phosphatase tensin homolog (6), LKB1 (7, 8), and even p53 (9). Such haploinsufficiency deviates from Knudson's model (10–12), suggesting that these particular tumor suppressors may succumb to inactivation processes that are distinct from genetic or epigenetic lesions (13). 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 (14, 15). That observation, along with the reported exceptions to Knudson's hypothesis (7, 8) and the precedent of lipids inactivating I{kappa}B kinase (16, 17), directed our attention to LKB1, a recently discovered tumor suppressor associated with Peutz-Jeghers hamartoma syndrome (18, 19). 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 (20–24). We report that reactive lipid species covalently modify LKB1 at a nucleophilic Cys210 residue in its activation loop, thereby inhibiting both the phosphorylation of AMPK{alpha}3 and the downstream propagation of signals through the LKB1-AMPK{alpha}-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 (25, 26).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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); CompleteTM protease inhibitor mixture and FuGENE 6 transfection reagent (Roche Applied Science); polyclonal antibodies directed against LKB1, phospho-Thr172-AMPK{alpha}, AMPK{alpha}, phospho-Ser79-ACC, ACC, phospho-Thr389-S6K, S6K, ERK, JNK, AKT, IKK{alpha}, IKK{gamma} and COX-2 (Cell Signaling Technologies); rapamycin (Cell Signaling Technologies); horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology); PVDF membranes and Western LightingTM 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 QuikChangeTM 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.


Figure 1
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  		FIGURE 1.
LKB1 signaling pathway. LKB1 (STK11) functions as an AMP-kinase kinase in cells. Activity of LKB1 is limited in cells with adequate ATP. Accumulation of AMP, or addition of its mimetic AICAR, activates the AMP-kinase kinase activity of LKB1, which converts AMPK{alpha} -> phospho-Thr172 AMPK{alpha}. In turn, phospho-Thr172 AMPK{alpha} converts ACC -> phospho-Ser79 acetyl-CoA carboxylase. Phospho-Thr172 AMPK{alpha} also phosphorylates TSC1/2, which inhibits mTOR-mediated conversion of S6K -> phospho-Thr389 S6K. Cells sense changes in their AMP/ATP ratio and use the LKB1-> AMPK{alpha} kinase cascade to maintain proper anabolic {leftrightarrow} catabolic homeostasis. LKB1 has a dual role as a tumor suppressor and metabolic regulator by antagonizing the phosphatidylinositol 3-kinase-AKT pathway, which propagates anabolic signals from insulin and proliferation signals from growth factors/oncogenes.

 
Isolation of Kinases Covalently Labeled by PG-Amidopentylbiotin—MCF-7 and RKO cells were treated with 10–60 µM PGA1-amidopentylbiotin or {Delta}12-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, 1x CompleteTM protease inhibitor, 2 mM sodium fluoride, and 2 mM sodium orthovanadate. The lysates were sonicated 10x for 1 s at 4 °C. After centrifugation at 10,000 x 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 x 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% beta-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), IKK{alpha} (1:1000), IKK{gamma} (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 LightingTM ECL reagents.

Transfection—LKB1-HA or LKB1 (C210S)-HA was transfected into RKO cells using 1 µg/µl DNA, 3 µl Lipofectamine2000TM for 20 h following the manufacturer's protocol. COX-2 was transfected into MCF-7 cells using 1 µg/µl DNA, 3 µl Lipofectamine2000TM 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 QuikChangeTM 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.

AMPK{alpha}, 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 (27), 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-AMPK{alpha} (1:1000), total AMPK{alpha} (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 LightingTM. The bands were analyzed using a Kodak Image Station 440TM, 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.


Figure 2
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  		FIGURE 2.
Covalent modification of cellular kinases by PGA1-amidopentylbiotin; comparison of LKB1 with other serine/threonine kinases. A shows the amino acid sequence alignment of the activation loop regions of JNK, ERK, p38, AKT, IKK, and LKB1 kinases. Cys179 in IKK corresponds with Cys210 in LKB1 (box and arrow). B shows immunoblots of total ERK, JNK, AKT, IKK, and LKB1 kinases in whole cell lysates (WC) from MCF-7 cells treated 4 h at 37 °C with 20 µM PGA1 (WC, lane 1), 20 µM PGA1-amidopentylbiotin (WC, lane 2), or 20 µM aminopentylbiotin (WC, lane 3). Whole cell lysates were incubated with neutravidin immobilized on agarose to sequester kinases with a biotin epitope, introduced de novo from their covalent reaction with PGA1-amidopentylbiotin. The immunoblots of kinases sequestered on neutravidin-agarose beads (NA pulldown) shows that only IKK and LKB1 contained a biotin epitope after incubation with PGA1-APB (NA pulldown, lane 2). None of the kinases contained a biotin epitope after incubation with PGA1 (NA pulldown, lane 1) or aminopentylbiotin (NA pulldown, lane 3), as controls.

 
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 2000TM 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Covalent Modification of Cellular LKB1 by Electrophilic Lipids—Cyclopentenone PG and 4-HNE react covalently with IKK (16, 17). 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 IKK{alpha} and IKKbeta. 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 {alpha},beta-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 (15). PGA1-amidopentylbiotin reacted covalently with cellular LKB1 and IKK{alpha} but not with ERK1/2, JNK2, AKT, or IKK{gamma} (Fig. 2B). These inert kinases lack the distinctive Cys residue corresponding to Cys179 in IKK{alpha} 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 {Delta}12-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 {Delta}12-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 AMPK{alpha} into phospho-Thr172-AMPK{alpha}. Basal levels of phospho-Thr172-AMPK{alpha} were low but detectable in MCF-7 cells grown in 10% FCS (Fig. 4A, lane 1). Phospho-Thr172-AMPK{alpha} 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-AMPK{alpha} by these stimuli (Fig. 4A, lanes 2, 4, and 6). Several different reactive lipid species that have electrophilic beta-carbons, including 4-HNE, 4-ONE, 15-deoxy-{Delta}12, {Delta}14-PGJ2, {Delta}12-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 {Delta}12-PGJ2 (n = 8), 3.0 ± 1.4 µM 4-HNE (n = 4), and 2.5 ± 1.0 µM 4-ONE (n = 3). The electrophilic beta-carbon appeared to be critical for inhibition, because unreactive lipids, including cyclopentenone PGB1, PGE2, PGF2{alpha}, 15-keto-PGF2{alpha}, and TxB2 at concentrations of 60–100 µM did not inhibit LKB1 activity (Fig. 4B).

Inhibition of the LKB1-AMPK{alpha}-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 AMPK{alpha}. For instance, PGA1, {Delta}12-PGJ2, and 4-HNE inhibited the phosphorylation of ACC, a substrate for phospho-Thr172-AMPK{alpha}, 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-AMPK{alpha} pathway with 2 mM AICAR reduced the level of phospho-Thr389-S6K, the active form of S6K (Fig. 5, B and C, lane 3). {Delta}12-PGJ2, alone, enhanced S6K activity in MCF-7 cells (Fig. 5, B and C, lane 2 versus lane 1). {Delta}12-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 {Delta}12-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 AMPK{alpha} pathway, {Delta}12-PGJ2 enhanced the distal formation of anabolically active phospho-Thr389-S6K, consistent with the scheme shown in Fig. 1.


Figure 3
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  		FIGURE 3.
Covalent modification of LKB1 involves its Cys210 residue. A, RKO cell transfected with Mock construct (lanes 1 and 2), HA-tagged LKB1 (lanes 3 and 4), or HA-tagged LKB1 C210S mutant (lanes 5 and 6) were treated with 60 µM PGA1 (lanes 1, 3, and 5) or PGA1-amidopentylbiotin (PGA1-APB)(lanes 2, 4 and 6) for 4 h at 37 °C. Whole cell lysates were incubated with neutravidin-agarose beads to isolate proteins containing a de novo biotin epitope (NA pulldown), or immunoprecipitated with immobilized goat anti-HA to estimate transfection efficiency (IP:HA). Immunoblots with anti-LKB1 antibody show that transfected RKO cells expressed HA-tagged wild type LKB1 (IP:HA lanes 3 and 4) and HA-tagged C210S mutant LKB1 (IP:HA, lanes 5 and 6). Only wild type LKB1 formed an adduct with PGA1-APB (NA pulldown, lane 4). Mutant LKB1 with a C210S substitution did not form an adduct (NA pulldown, lane 6). B, immunoblot of wild type LKB1 expressed constitutively in MCF-7 cells treated for 4 h at 37 °C with 60 µM of PGA1 (WC, lane 1), PGA1-APB (WC, lane 2), {Delta}12-PGJ2, (WC, lane 3), or {Delta}12-PGJ2-APB (WC, lane 4). Whole cell lysates (WC) were incubated with neutravidin-agarose beads to isolate proteins containing a biotin epitope. Immunoblots show that LKB1 formed an adduct with PGA1-APB (NA pulldown, lane 2) and {Delta}12-PGJ2-APB (NA pulldown, lane 4).

 
Inhibition of LKB1 Activity by Ectopic Expression of Cyclooxygenase-2—COX-2 expression and activity are increased in inflammation and in tumors (25, 26). 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-AMPK{alpha} 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-AMPK{alpha} 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-AMPK{alpha} 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-AMPK{alpha} 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 (28), which accounts for PGE2 and PGD2 biosynthesis by mock-transfected cells.


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  		TABLE 1
Prostaglandin formation by MCF-7 cells

Values are mean ± S.E., n = 3 experiments. MCF-7 cells were transfected with a COX-2 or a mock construct. 24 hours after transfection, cells were incubated for 1 h with culture medium containing vehicle or 10 µM NS-398. Cells were then incubated with 100 µM arachidonic acid (AA) for 30 min, and levels of PGE2 and PGD2 in the medium were quantified by immunoassay.

 


Figure 4
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  		FIGURE 4.
Lipids with electrophilic beta-carbons inhibit serine/threonine kinase activity of cellular LKB1. A, MCF-7 cells were treated for 6 h with medium containing 10% serum (lanes 1–4) or 1% serum (lanes 5 and 6) and then incubated with PGA1 (lanes 2, 4, and 6) or vehicle control (lanes 1, 3, and 5) for 4 h at 37 °C, followed by 30 min with vehicle or 2 mM AICAR (lanes 3 and 4). Immunoblots of total AMPK{alpha} and phospho-Thr172 AMPK{alpha} show that PGA1 inhibits the serine/threonine kinase activity of cellular LKB1. B, histogram of LKB1 activity in MCF-7 cells treated with 60 µM of each designated lipid for 4 h, followed by 2 mM AICAR treatment for 30 min. The samples were lysed, fractionated by SDS-PAGE, and their phospho-Thr172 AMPK{alpha} and total AMPK{alpha} were determined by Western immunoblot. The values represent the net band intensity of phospho-Thr172 AMPK{alpha} (mean ± S.E., n = 3). LKB1 activity in MCF-7 cells treated with PGB1, PGF2{alpha}, 15-keto-PGF2{alpha}, TxB2, or PGE2 was statistically indistinguishable from the vehicle-treated control. LKB1 activity was inhibited only in cells treated with PGA1, {Delta}12-PGJ2, 15-deoxy-{Delta}12, {Delta}14-PGJ2, 4-HNE, or 4-ONE (p < 0.05, analysis of variance with Bonferroni's post-hoc test). These structure-activity data suggest an inactivation mechanism involving Michael addition of the nucleophilic Cys210 residue LKB1 to lipids with electrophilic beta-carbons. C, representative immunoblot showing dose-dependent inhibition of AMPK phosphorylation in MCF-7 cells treated 4 h with PGA1, 4-HNE, and 4-ONE or Me2SO (0.10%) followed by 2 mM AICAR.

 


Figure 5
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  		FIGURE 5.
Electrophilic lipids inhibit phosphorylation of acetyl-CoA carboxylase and S6 kinase downstream from their inhibition of LKB1 -> AMP kinase. A, immunoblot of total ACC and phospho-Ser79-ACC in lysates from MCF-7 cells incubated with 60 µM PGB1, PGA1, {Delta}12-PGJ2, 4-HNE, or Me2SO (0.10%) vehicle control for 4 h and then with 2 mM AICAR for 30 min to stimulate the LKB1 -> AMPK{alpha} signaling pathway and subsequent ACC phosphorylation. Representative lipids that inhibited LKB1, at the apex of the signaling pathway, also attenuated the phosphorylation of ACC, a substrate of AMPK{alpha} (Fig. 1). B, immunoblot of total S6K and phospho-Thr389 S6K in lysates of MCF-7 cells incubated 4 h with vehicle (lane 1); 4 h with 60 µM {Delta}12-PGJ2 alone (lane 2); 30 min with 2 mM AICAR alone (lane 3); 4 h with {Delta}12-PGJ2 and then 30 min with AICAR (lane 4); 2 h with 50 nM rapamycin alone (lane 5); or 4 h with {Delta}12-PGJ2, plus 2 h with 50 nM rapamycin, plus 30 min with AICAR (lane 6). C, histogram of data in B. The intensity of the S6K and phospho-Thr389 S6K bands was measured and depicted as a percentage of the control (i.e. 100% = vehicle alone, lane 1). Values represent mean ± S.E., n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, LKB1 was identified as the gene responsible for Peutz-Jeghers syndrome (18, 19), 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 (29), 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 (20–24).

LKB1 has two vital cellular roles as follows: tumor suppressor and regulator of anabolic/catabolic homeostasis. LKB1 achieves its purpose by negatively regulating the phosphatidylinositol 3-kinase-AKT pathway (Fig. 1). 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) (23). 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 (30–32). Low expression of LKB1 in breast tumors is associated with poor prognosis and survival (33) and with the transition from pre-malignant to malignant tumor growth in lung cancer (34). Most interestingly, several studies show that hamartomas overexpress COX-2 (35, 36), and its expression facilitates tumorigenesis via a hamartoma-adenoma-carcinoma sequence (37, 38). Although speculative at this point, our results suggest that inhibition of LKB1 because of its chemical inactivation by reactive lipid species or overexpression of COX-2, such as in Fig. 6, may have consequences similar to loss of expression of LKB1 or mutational inactivation.


Figure 6
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  		FIGURE 6.
Inhibition of cellular LKB1 activity by ectopically expressed cyclooxygenase-2. A, histogram depicting the relative amount of phospho-Thr172 AMPK{alpha} in MCF-7 cells transfected with a mock construct ({blacksquare}) or COX-2 ({square}) and treated with (R)-ibuprofen (Ibu). 24 hours after transfection, cells were incubated for 30 min with medium containing 10% FCS with 100 µM (R)-ibuprofen or vehicle control followed by 4 h of incubation with 100 µM arachidonic acid (AA). 2 mM AICAR was added to all cells to stimulate LKB1 activity. Cells expressing COX-2 and treated with 100 µM arachidonic acid showed a decrease in LKB1 activity as measured by phospho-AMPK{alpha} content relative to control. Cells expressing COX-2 and exposed to 100 µM (R)-ibuprofen showed a significant recovery in LKB1 activity as measured by phospho-AMPK{alpha} compared with cells not treated with ibuprofen. B, representative immunoblot showing inhibition of the phosphorylation of AMPK{alpha} because of COX-2 transfection and arachidonic acid treatment and recovery of the phosphorylation of AMPK{alpha} because of preincubation with ibuprofen. The intensity of the AMPK{alpha} and phospho-Thr172 AMPK{alpha} bands was measured and depicted as a percentage of the control (i.e. 100% = AICAR alone, lane 1). Values represent mean ± S.E., n = 3.

 
LKB1 is a tumor suppressor that often retains one wild type allele, deviating from Knudson's two-hit hypothesis. 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 (16, 39). Based on site-directed mutagenesis and pharmacological experiments with inert and reactive eicosanoids, Rossi et al. (16) proposed a Michael addition at the Cys179 residue in the activation loop as the molecular basis for inhibition of IKK{alpha} and IKKbeta. Because LKB1 is a rare example of a tumor suppressor, which functions as a serine/threonine kinase (18, 19), we compared the amino acid sequence of LKB1 flanking its activation loop with those for IKK, ERK, JNK, AKT, and p38. This sequence alignment revealed a Cys210 residue in LKB1 that corresponded to the Cys179 residue in IKK. These Cys residues are distinct to LKB1, IKK{alpha}, and IKKbeta but not ERK, JNK, and p38 kinases, which have Met or Leu in comparable positions (Fig. 2). Our results show that cyclopentenone PGs of the A- and J-series covalently modify LKB1 and IKK{alpha} but not ERK1/2, JNK2, IKK{gamma}, AKT, or an LKB1 mutant with Cys210 replaced by Ser210. Thus, these cellular serine/threonine kinases do not react indiscriminately with PGA1-amidopentylbiotin under our experimental conditions. The results in Figs. 2 and 3 are compatible with the following: (i) the pharmacological data mentioned above (16, 39); (ii) the known chemical reactivity between thiols (–SH) and {alpha},beta-unsaturated carbonyls (Michael reaction); and (iii) the influence of other amino acids on steric accessibility of the thiol and the chemical reactivity of cysteine. Interactions between cysteine and other amino acids determine the pKa and nucleophilic character of its thiol (RSH {leftrightarrows} RS–). Virtually all lipids bind to cellular proteins by reversible, low affinity interactions (Kd ~ 10–6 liter/mol). For some lipids with an {alpha},beta-unsaturated carbonyl, this low affinity interaction with proteins seems to position their electrophilic beta-carbon near a nucleophilic substituent that is favorably disposed for Michael addition, i.e. Cys179 in IKK (16), Cys210 in LKB1, Cys38 in the p65 subunit of NF{kappa}B (40), Cys62 in the p50 unit of NF{kappa}B (41), Cys29 in cathepsin (42), and Cys35 in thioredoxin (43), and results in a covalent lipid adduct. Thus, pKa and steric accessibility of cysteinyl thiols strongly influence, and limit, which particular cellular proteins can form Michael adducts with A- or J-series PGs (15, 16, 40, 41, 43, 44) or 4-HNE (15, 17, 42, 45).

The chemistry of nucleophilic Michael addition reactions argues against any individual {alpha},beta-unsaturated carbonyl acting as a regulatory molecule via specific, covalent reaction with any individual protein receptor. If these reactive lipids modulate biological processes, they probably do so with less specificity than classical autocoids. For example, multiple lipids with {alpha},beta-unsaturated ketones can react with a particular protein, e.g. thioredoxin reductase (15), IKK (16), and LKB1. Likewise, a particular electrophilic lipid, e.g. 4-HNE, can react with more than one protein (15, 17, 42, 45). During oxidative stress, inflammation, or overexpression of COX-2, the biological milieu may contain multiple electrophilic species. Consequently, each species with an {alpha},beta-unsaturated ketone will contribute, stoichiometrically, to any pathophysiological effect. 4-HNE, cyclopentenone PGs, and 8-iso-cyclopentenone PGs, have been detected in vivo within cellular proteins at sites of inflammation (46–49). Investigators have even hypothesized that these reactive lipid species help resolve inflammation by covalently modifying I{kappa}B kinases and repressing NF{kappa}B (16, 50). Such an unconventional mechanism of action for resolving inflammation may confer inevitable risks, such as the impairment of other proteins and tumors suppressors. Disruption of tumor suppressors by inflammatory mediators seems especially relevant to cancers associated with chronic inflammation or overexpression of cyclooxygenase and lipooxygenase (25, 26).


   FOOTNOTES
 
* This work was supported in part by United States Public Health Services Grants R01 AI26730 and PO1 CA73992 and the Huntsman Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a predoctoral fellowship from the American Foundation for Pharmaceutical Education. Back

2 Holds the Dee Glenn and Ida W. Smith Chair for Cancer Research. To whom correspondence should be addressed: Dee Glenn and Ida W. Smith Chair of Cancer Research, Huntsman Cancer Institute, 2000 Circle of Hope, University of Utah, Salt Lake City, UT 84112-5550. Tel.: 801-581-6204; Fax: 801-585-0011; E-mail: frank.fitzpatrick{at}hci.utah.edu.

3 The abbreviations used are: AMPK{alpha}, AMP-activated kinase {alpha}; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; APB, amidopentyl biotin; 4-HNE, 4-hydroxy-2-nonenal; IKK, I{kappa}B kinase; NF{kappa}B, nuclear factor {kappa}B; 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tomi Makela for providing the plasmids encoding LKB1.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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