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Evidence That Phosphatidylinositol 3-Kinase- and Mitogen-activated Protein Kinase Kinase-4/c-Jun NH2-terminal Kinase-dependent Pathways Cooperate to Maintain Lung Cancer Cell Survival*

Open AccessPublished:April 24, 2003DOI:https://doi.org/10.1074/jbc.M300997200
      Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85α regulatory subunit of PI3K (Δp85) induced apoptosis. Whereas PTEN and Δ85 both inhibited activation of AKT/protein kinase B, only Δp85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Δp85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.
      Class I phosphatidylinositol 3-kinase (PI3K)
      The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-bisphosphate; PI(4,5)P, phosphatidylinositol 4,5-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; MKK, mitogenactivated protein kinase kinase; JNK, c-Jun NH2-terminal kinase; NSCLC, non-small cell lung cancer; MEF, mouse embryo fibroblast; EGF, epidermal growth factor; IGF-1, insulin-like growth factor-1; MAP, mitogen-activated protein kinase; SH2, Src homology domain 2; HA, hemagglutinin; PBD, p21 binding domain; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, myelin basic protein; GSK3, glycogen synthase kinase 3; CDK2, cyclin-dependent kinase 2; ERK1/2, extracellular signal-regulated kinase 1/2; BrdUrd, bromodeoxyuridine; PTEN, phosphatase and tensin homolog deleted from chromosome 10.
      1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-bisphosphate; PI(4,5)P, phosphatidylinositol 4,5-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; MKK, mitogenactivated protein kinase kinase; JNK, c-Jun NH2-terminal kinase; NSCLC, non-small cell lung cancer; MEF, mouse embryo fibroblast; EGF, epidermal growth factor; IGF-1, insulin-like growth factor-1; MAP, mitogen-activated protein kinase; SH2, Src homology domain 2; HA, hemagglutinin; PBD, p21 binding domain; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, myelin basic protein; GSK3, glycogen synthase kinase 3; CDK2, cyclin-dependent kinase 2; ERK1/2, extracellular signal-regulated kinase 1/2; BrdUrd, bromodeoxyuridine; PTEN, phosphatase and tensin homolog deleted from chromosome 10.
      consists of a family of heterodimeric complexes composed of a p110 catalytic subunit and a regulatory subunit that exists predominantly in a p85 form (
      • Toker A.
      • Cantley L.C.
      ,
      • Vanhaesbrock B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Wymann M.P.
      • Pirola L.
      ). The known gene family members for p85 (α, β, and γ) and p110 (α, β, δ, and γ) are expressed in a tissue-specific fashion. p85α and -β can also exist in smaller forms (p50 and p55). PI3K phosphorylates the D3 position of PI on PI(4)P and PI(4,5)P to produce PI(3,4)P2 and PI(3,4,5)P3. The 3′ sites of PI(3,4)P2 and PI(3,4,5)P3 are dephosphorylated by the PTEN tumor suppressor, whereas the 5′ site of PI(3,4,5)P3 is dephosphorylated by SHIP to produce PI(3,4)P2 (
      • Toker A.
      • Cantley L.C.
      ). These mechanisms tightly regulate the levels of 3-phosphorylated PI in the cell. PI(3,4,5)P3 and PI(3,4)P2 recruit the pleckstrin homology domains of specific intracellular proteins to the plasma membrane, an essential event in the activation of PI3K-dependent kinases such as phosphoinositide-dependent kinase-1 and AKT, also known as protein kinase B. In addition, AKT phosphorylation at Thr308 by phosphoinositide-dependent kinase-1 and Ser473 by integrin-linked kinase (and possibly other kinases) constitutes an essential event in AKT activation (
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      • James S.R.
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      ).
      The PI3K pathway clearly has a key role in cellular survival and transformation. AKT phosphorylates several pro- and anti-apoptotic proteins, including the Bcl-2 family member BAD, caspase-9, cyclic AMP response element-binding protein, the inhibitor of NF-κB kinase IKKα, and forkhead transcription factor-1 (
      • Di Cristofano A.
      • Pandolfi P.P.
      ). Tumor cells feature genetic and epigenetic alterations of p85α, p110α/β, AKT2, AKT3, and PTEN that activate PI3K-dependent signaling (
      • Bellacosa A.
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      • Wan M.
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      • Cheng J.Q.
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      • Testa J.R.
      ,
      • Janssen J.W.G.
      • Schleithoff L.
      • Bartram C.R.
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      ,
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypanina K.
      • Bose S.
      • Wang S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Shayesteh L.M.
      • Lu Y.
      • Kuo W.L.
      • Baldocchi R.
      • Godfrey T.
      • Collins C.
      • Pinkel D.
      • Powell B.
      • Mills G.B.
      • Gray J.W.
      ,
      • Steck P.A.
      • Pershouse M.A.
      • Jasser S.A.
      • Yung W.K.A.
      • Lin H.
      • Ligon A.H.
      • Langford L.A.
      • Baumgard M.L.
      • Hattier T.
      • Davis T.
      • Frye C.
      • Hu R.
      • Swedlund B.
      • David H.F.
      • Tavtigian T.
      • Tavtigian S.V.
      ,
      • Teng D.H.
      • Hu R.
      • Lin H.
      • Davis T.
      • Iliev D.
      • Frye C.
      • Swedlund B.
      • Hansen K.L.
      • Vinson V.L.
      • Gumpper K.L.
      • Ellis L.
      • El-Naggar A.
      • Frazier M.
      • Jasser S.
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      • Lee J.
      • Mills G.B.
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      • Tornos C.
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      • Yung W.K.
      • Fujii G.
      • Berson A.
      • Steck P.A.
      ). In vitro studies have confirmed the oncogenic effects of PI3K and its downstream mediators as well as the tumor-suppressive properties of PTEN (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ,
      • Chang H.W.
      • Aoki M.
      • Fruman D.
      • Auger K.R.
      • Bellacosa A.
      • Tsichlis P.N.
      • Cantley L.C.
      • Roberts T.M.
      • Vogt P.K.
      ,
      • Furnari F.B.
      • Lin H.
      • Huang H.-J.
      • Cavenee W.K.
      ,
      • Klippel A.
      • Escobedo M.A.
      • Wachowicz M.S.
      • Apell G.
      • Brown T.W.
      • Giedlin M.A.
      • Kavanaugh W.M.
      • Williams L.T.
      ,
      • Li D.M.
      • Sun H.
      ,
      • Lu Y.
      • Lin Y.Z.
      • LaPushin R.
      • Cuevas B.
      • Fang X.
      • Yu S.X.
      • Davies M.A.
      • Khan H.
      • Furui T.
      • Mao M.
      • Zinner R.
      • Hung M.C.
      • Steck P.
      • Siminovitch K.
      • Mills G.B.
      ).
      PI3K mediates its oncogenic effects, in part, through the GTP-binding protein Rac-1, which plays a key role in the reorganization of the actin cytoskeleton induced by growth factors or oncogenic Ras (
      • Qiu R.G.
      • Chen J.
      • Kirn D.
      • McCormick F.
      • Symons F.
      ). p85α interacts directly with Rac-1 (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Ras activates Rac-1 indirectly as a consequence of PI3K-mediated phosphorylation of membrane PIs (
      • Nimnual A.S.
      • Yatsula B.A.
      • Bar-Sagi D.
      ). PI(3,4,5)P3 binds to the guanosine nucleotide-exchange factor SOS, stimulating SOS to load Rac-1 with GTP, an essential event in Rac-1 activation. Rac-1, in turn, activates downstream signaling through PAK-1 and its mediators, which include mitogen-activated protein kinase kinase-4 (MKK4) and its substrates c-Jun NH2-terminal kinase (JNK) and p38/HOG1 (
      • Davis R.J.
      ).
      Certain cancer cell types with PTEN gene loss have constitutively active PI3K and undergo apoptosis in response to pharmacologic or genetic inhibition of PI3K (
      • Neshat M.S.
      • Mellinghoff I.K.
      • Tran C.
      • Stiles B.
      • Thomas G.
      • Petersen R.
      • Frost P.
      • Gibbons J.J.
      • Wu H.
      • Sawyers C.L.
      ). Most non-small cell lung cancer (NSCLC) cell lines demonstrate hallmarks of PI3K pathway activation, such as phosphorylation of AKT and its downstream mediators, but have a wild-type PTEN gene (
      • Brognard J.
      • Clark A.S.
      • Ni Y.
      • Dennis P.A.
      ,
      • Massion P.
      • Kuo W.L.
      • Stokoe D.
      • Olshen A.B.
      • Treseler P.A.
      • Chin K.
      • Chen C.
      • Polikoff D.
      • Jain A.N.
      • Pinkel D.
      • Albertson D.G.
      • Jablons D.M.
      • Gray J.W.
      ,
      • Forgacs E.
      • Biesterveld E.J.
      • Sekido Y.
      • Fong K.
      • Muneer S.
      • Wistuba I.I.
      • Milchgrub S.
      • Brezinschek R.
      • Virmani A.
      • Gazdar A.F.
      • Minna J.D.
      ,
      • Moore S.M.
      • Rintoul R.C.
      • Walker T.R.
      • Chilvers E.R.
      • Haslett C.
      • Sethi T.
      ,
      • Yokomizo A.
      • Tindall D.J.
      • Drabkin H.
      • Gemmill H.
      • Franklin W.A.
      • Yang P.
      • Sugio K.
      • Smith D.I.
      • Liu D.
      ,
      • Soria J.C.
      • Lee H.Y.
      • Lee J.I.
      • Wang L.
      • Issa J.P.
      • Kemp B.L.
      • Liu D.D.
      • Kurie J.M.
      • Khuri F.R.
      ). Despite having wild-type PTEN, NSCLC cells undergo apoptosis in response to PI3K pathway inhibition (
      • Brognard J.
      • Clark A.S.
      • Ni Y.
      • Dennis P.A.
      ). The apoptosis reported by Brognard et al. (
      • Brognard J.
      • Clark A.S.
      • Ni Y.
      • Dennis P.A.
      ) may depend in part on the absence of serum, which rescues cells from apoptosis induced by PI3K inhibition (
      • Li D.M.
      • Sun H.
      ,
      • Lu Y.
      • Lin Y.Z.
      • LaPushin R.
      • Cuevas B.
      • Fang X.
      • Yu S.X.
      • Davies M.A.
      • Khan H.
      • Furui T.
      • Mao M.
      • Zinner R.
      • Hung M.C.
      • Steck P.
      • Siminovitch K.
      • Mills G.B.
      ,
      • Lin J.
      • Adam R.M.
      • Santiestevan E.
      • Freeman M.R.
      ,
      • Furnari F.B.
      • Huang H.J.
      • Cavenee W.K.
      ). Thus, serum-induced activation of other peptide growth factor-induced signaling pathways can overcome the pro-apoptotic effect of PI3K inhibition. In this study, we investigated the signaling pathways that interact with PI3K to control NSCLC cell survival. Using pharmacologic and genetic approaches, we found that inhibition of PI3K-dependent signaling alone induced proliferative arrest, whereas inhibition of both PI3K and MKK4/JNK-dependent pathways induced apoptosis. These findings indicate that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.

      EXPERIMENTAL PROCEDURES

      Reagents—H358, H661, Calu-6, H460, H226B, H226Br, H441, and H1299 NSCLC cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (complete medium). COS-7 cells and MKK4-null and wild-type mouse embryo fibroblast (MEF) cells (
      • Nishina H.
      • Fischer K.D.
      • Radvani L.
      • Shahinian A.
      • Hakem R.
      • Rubie E.A.
      • Bernstrin A.
      • Mak T.W.
      • Woodgett J.R.
      • Penninger J.M.
      ) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. We purchased epidermal growth factor (EGF) (Invitrogen), insulin-like growth factor-1 (IGF-1) (R&D Systems, Minneapolis, MN), the class I PI3K inhibitor LY294002 (Calbiochem, La Jolla, CA), tumor necrosis factor-α (Sigma), recombinant GST-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), myelin basic protein (MBP) (New England Biolabs, Beverly, MA), GST-GSK3β (Santa Cruz), and protein A-G-agarose beads (Santa Cruz). We also purchased rabbit polyclonal antibodies against human phospho-AKT (pAKT1; Ser473) and AKT1 (New England Biolabs), phospho-JNK (Thr183/Tyr185; Cell Signaling Technologies), p85α, cyclin-dependent kinase (CDK) 2, and p27 (Santa Cruz), and murine monoclonal antibodies against human PTEN (Santa Cruz), phosphoextracellular signal-regulated kinase (ERK) (Thr202/Tyr204; Cell Signaling), caspase-3 and -9 (BD Pharmingen), poly(ADP-ribose) polymerase (VIC5) (Roche Diagnostics), and goat polyclonal antibodies against human ERK1/2, JNK-1, and β-actin (Santa Cruz). The adenoviral vector expressing wild-type p85α (Adex1CAp85α-HA) has been described elsewhere (
      • Ueki K.
      • Algenstaedt P.
      • Mauvais-Jarvis F.
      • Kahn C.R.
      ). A recombinant adenovirus expressing human PTEN under the control of a cytomegalovirus (CMV) promoter was a gift from Dr. W. K. A. Yung (M. D. Anderson Cancer Center). Plasmid expression vectors containing Rac-1 (Val12) and MKK1 (R4F) were gifts from Dr. Melanie Cobb (The University of Texas Southwestern Medical Center, Dallas, TX).
      Generation of Ad5-Δp85—Δp85 is a bovine p85α mutant lacking 35 amino acids (residues Met479 to Lys513) in the inter-SH2 region that are necessary for binding to the p110 catalytic subunit (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Waterfield M.D.
      • Kasuga M.
      ). The Δp85 cDNA was inserted into the 5′ end of the bovine growth hormone polyadenylation signal at the HindIII site of the pAd-shuttle vector, which was a gift from Dr. Jack Roth (M. D. Anderson Cancer Center). The Δp85-containing shuttle vector was digested with BstI/ClaI and inserted into the pAd-speed vector (
      • Ji L.
      • Nishizaki M.
      • Gao B.
      • Burbee D.
      • Toyooka S.
      • Kamibayashi C.
      • Xu K.
      • Yen N.
      • Atkinson E.N.
      • Fang B.
      • Lerman M.I.
      • Roth J.A.
      • Minna J.D.
      ). 293 cells were transfected with the resulting plasmid and then maintained until the onset of the cytopathic effect. Viral titers were determined by plaque assays and spectrophotometric analysis. The presence of Δp85 in viral particles was confirmed by dideoxy-DNA sequencing and Western blot analysis.
      Cell Growth Assays—NSCLC cell lines were seeded at 1–2 × 103 cells/well in 96-well plates. After 24 h, cells were incubated in serum-free conditions with 5 × 102, 1 × 103, 5 × 103, or 1 × 104 p/cell of Ad5-Δp85, Ad5-PTEN, or Ad5-CMV (control virus). After 2 h, cells were changed to complete medium. In the case of LY294002 treatment, cells were treated with 0.2, 2, 20, 40, 60, or 80 μm LY294002 in complete medium, which was changed every 48 h. After 5 days, cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
      Western Blot Analysis—Whole cell lysates were prepared by incubating cell pellets in lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 0.2 mm EGTA, 1% Nonidet P-40, 10% glycerol, 1mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 mm sodium fluoride, 5 mm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mm benzamidine) for 20 min on ice. After clarification by centrifugation at 13,000 × g for 20 min, the supernatants were collected, and the protein concentration was determined with a BCA protein assay kit (Pierce). Cell lysates (30 μg) were subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride nitrocellulose membrane (Bio-Rad). Membranes were immunoblotted overnight at 4 °C with primary antibodies in Tris-buffered saline containing 5% nonfat dry milk. Antibody binding was detected with an electrochemiluminescence kit (Amersham Biosciences) according to the manufacturer's directions.
      Cell Cycle and Apoptosis Assays—For these experiments, 1 × 106 H1299 cells were transferred onto 100-mm plates. Twenty-four hours later, the cells were incubated with 1 × 103,5 × 103,or1 × 104 particles of Ad5-Δp85 or Ad5-PTEN per cell. For combination treatments, H1299 cells were transiently transfected with 5 μg of plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector using FuGENE (Roche Diagnostics). After 6 h, the cells were incubated for 2 h in serum-free conditions with Ad5-Δp85 or Ad5-CMV at 1 × 103 or 5 × 103 particles/cell. Cells were allowed to grow in complete medium for 48 h before being subjected to apoptosis assays.
      Apoptosis and cell cycle progression were measured by TUNEL with the APO-BRDU staining kit (Phoenix Flow Systems, San Diego, CA). Floating cells and attached cells were dispersed with trypsin-EDTA, pelleted, washed, and fixed in 1% paraformaldehyde for 15 min on ice and then fixed in 70% ethanol. The fixed cells were washed and incubated with DNA labeling solution containing terminal deoxynucleotidyltransferase reaction buffer, deoxynucleotidyltransferase enzyme, and bromodeoxyuridine triphosphate (BrdUrd-dUTP). The cells were rinsed before being resuspended with fluorescein-PRB-1 antibody solution and analyzed by flow cytometry in the presence of propidium iodide/RNase solution. Analyses of 3,000 to 10,000 events were done with a FACScan flow cytometer (BD Pharmingen) equipped with a 488-nm argon ion laser and two software packages: CellQuest 3.1 (BD Pharmingen) and ModFit LT 2.0 (Verity Software House, Topsham, ME). Live gating of the forward and orthogonal scatter channels was used to exclude debris and to selectively acquire cell events. A dual display of DNA area (linear red fluorescence) and BrdUrd-dUTP incorporation (FITC-PRB-1) was used to determine the percentage of propidium iodine-stained cells that were apoptotic.
      Apoptosis was also determined by the detection of nucleosomal DNA fragmentation by using the TACS apoptotic DNA laddering kit (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer's protocol. Briefly, DNA was isolated from cells after adenovirus transfection or LY294002 treatment by incubating them in lysis buffer. DNA samples were subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
      Immune Complex Kinase Assay—H1299 cells were incubated for 2 h with Ad5-CMV, Ad5-Δp85, or Ad5-PTEN at 1 × 103, 5 × 103, or 1 × 104 p/cell in serum-free conditions, changed to complete medium, and incubated for 48 h. Cells were then washed twice in 1× phosphate-buffered saline, serum-starved for 24 h, treated with 50 ng/ml EGF for 15 min, and lysed in lysis buffer. Extracts were subjected to immunoprecipitation (100 μg) with antibodies to JNK1, AKT1/2, or ERK1/2 by rotation at 4 °C overnight. Protein A-G-agarose beads (20 μl) were added, and the solution was incubated at 4 °C for 1 h. The beads were washed three times with lysis buffer and once with kinase buffer (20 mm Hepes (pH 7.5), 20 mm β-glycerol phosphate, 10 mm MgCl2, 1 mm dithiothreitol, and 50 mm sodium orthovanadate). Kinase assays were performed by incubating the beads with 30 μl of kinase buffer, to which 20 μm cold ATP, 5 μCi of [γ-32P]ATP (2,000 cpm/pmol), and 2 μg of GST-c-Jun, GST-GSK3β, or MBP as substrates were added. The kinase reaction was performed at 30 °C for 20 min. The samples were then suspended in 1× Laemmli buffer and boiled for 5 min, and the samples were analyzed by 12% SDS-PAGE. The gel was dried and autoradiographed.
      Immune complex assays were also performed with COS-7 cells, which were transiently transfected for 6 h with 5 μg of plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector using FuGENE. The cells were then transfected with Ad5-Δp85 or Ad5-CMV (1 × 103 or 5 × 103 particles/cell) and incubated in complete medium for 24 h. The cells were then changed to serum-free medium for 24 h, treated with IGF-1 (50 ng/ml) for 15 min, and lysed. JNK and ERK were immunoprecipitated from 100 μg of total cell lysates and subjected to kinase assays using GST-c-Jun and MBP, respectively, as substrates.
      Rac-1 Activity Assays—Pull-down assays with GST-tagged p21 binding domain (PBD) of PAK-1 were performed as follows. COS-7 cells were co-transfected with 2 μg of HA-tagged p85α, HA-tagged p110α, and Δp85 (2, 4, or 6 μg) using LipofectAMINE (Invitrogen). Total amount of DNA transfected per plate was equalized with empty vector. After 6 h, transfectants were washed and changed to normal growth medium. After 24 h, transfectants were serum-starved for 16 h, treated with 50 ng/ml EGF or IGF-1 for 15 min, and lysed. PAK-1 PBD-agarose (5 μg in a 50% slurry) was added to the lysates and the mixture was incubated for 1 h at 4 °C. The bead pellet was collected by centrifugation (5 s at 14,000 × g) and the supernatant was drained off. The beads were then washed and suspended in 20 μl of 1× Laemmli sample buffer. Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted against Rac-1 and CDC42 polyclonal antibodies.

      RESULTS

      PI3K-dependent Pathway Contributes to NSCLC Cell Proliferation and Survival—We investigated the effects of PI3K inhibition on the proliferation and viability of H1299 NSCLC cells, which have a wild-type PTEN gene (
      • Forgacs E.
      • Biesterveld E.J.
      • Sekido Y.
      • Fong K.
      • Muneer S.
      • Wistuba I.I.
      • Milchgrub S.
      • Brezinschek R.
      • Virmani A.
      • Gazdar A.F.
      • Minna J.D.
      ). H1299 cells were transfected with recombinant adenoviruses that express PTEN (Ad5-PTEN) or Δp85 (Ad5-Δp85), a p85α dominant-negative mutant lacking the inter-SH2 residues required for binding to the p110 catalytic domain (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Waterfield M.D.
      • Kasuga M.
      ). Transfection of H1299 cells with Ad5-PTEN or Ad5-Δp85 increased the expression of the adenoviral gene products and suppressed pAKT levels (Fig. 1), providing evidence that these adenoviral vectors effectively blocked PI3K-dependent signaling. When H1299 cells were incubated with Ad5-PTEN or Ad5-Δp85, cell number decreased in a dose-dependent fashion (Fig. 2, A and B). H1299 cell number also decreased in a dose-dependent manner after treatment with LY294002, a competitive inhibitor of ATP binding to all isoforms of class I PI3K (Fig. 2C). Other NSCLC cell lines with wild-type PTEN (H358, Calu-6, H460, H661, H226B, H441, H1299, and H226Br) underwent a similar decrease in cell number following treatment with LY294002 or transfection with Ad5-Δp85 or Ad5-PTEN (data not shown).
      Figure thumbnail gr1
      Fig. 1Expression of adenoviral gene products and pAKT (Ser473) in H1299 NSCLC cells after transfection with Ad5-CMV, Ad5-Δp85, or Ad5-PTEN. H1299 cells were treated with medium alone (–) or incubated with the indicated viral particles (particles/cell). Three days later, the cells were lysed, and 30 μg of whole cell lysates were subjected to Western blot analysis of PTEN, p85α, pAKT (Ser473), and, as a control, total AKT1 (AKT1).
      Figure thumbnail gr2
      Fig. 2Effect of PI3K inhibition on H1299 cell numbers. H1299 cells were (A) incubated with the indicated titers of Ad5-PTEN or Ad5-CMV, (B) incubated with the indicated titers of Ad5-Δp85 or Ad5-CMV, or (C) treated with medium alone (0) or the indicated doses of LY294002. The cells were incubated for 5 days, at which time they were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Results are expressed relative to the density of cells treated with medium alone. Each value is the mean (± S.D.) of five identical wells.
      We next investigated whether PI3K inhibition induced proliferative arrest or apoptosis of NSCLC cells by performing flow cytometric analysis of H1299 cells transfected with Ad5-PTEN or Ad5-Δp85 and then stained with propidium iodide (Fig. 3A). Ad5-PTEN transfection induced proliferative arrest in the G0/G1 phase of the cell cycle, with minimal evidence of programmed cell death, as shown by the lack of a hypodiploid peak. Although Ad5-Δp85 transfection also caused an accumulation of cells in G1, its most striking effect was apoptosis, as indicated by the appearance of a hypodiploid peak. We examined this finding further by using terminal deoxynucleotidetransferase nick-end labeling (TUNEL), a more sensitive assay for apoptosis, and found low levels of DNA fragmentation in cells transfected with Ad5-PTEN (Fig. 3B). In contrast, transfection with Ad5-Δp85 produced much more DNA fragmentation, which is compatible with the induction of high levels of apoptosis (Fig. 3B).
      Figure thumbnail gr3
      Fig. 3Evidence of proliferative arrest and apoptosis in cells incubated with Ad5-PTEN or Ad5-Δp85. H1299 cells were incubated for 3 days with the indicated titers of Ad5-PTEN, Ad5-Δp85, or Ad5-CMV. Floating and adherent cells were isolated, fixed with 1% paraformaldehyde and 70% ethanol, stained with propidium iodide, and subjected to flow cytometric analysis to determine (A) the percentages of cells in specific phases of the cell cycle (pre-G1, G1, S, and G2/M) and (B) percentages that were apoptotic (TUNEL analysis). Results are summarized in the adjoining tables.
      We investigated the effect of Ad5-PTEN and Ad5-Δp85 on signaling events known to contribute to apoptosis, proliferative arrest, or both (Fig. 4). Ad5-Δp85 transfection reduced the levels of procaspase-9, procaspase-3, and poly(ADP-ribose) polymerase, demonstrating evidence of caspase activation and proteolysis of a caspase-3 substrate. In contrast, Ad5-PTEN transfection decreased CDK2 levels and increased p27 CDK inhibitor levels without evidence of caspase activation or poly-(ADP-ribose) polymerase cleavage. Together, these findings support a role for PI3K in the proliferation of NSCLC cells and demonstrate a pro-apoptotic effect of Δp85.
      Figure thumbnail gr4
      Fig. 4Effects of Ad5-PTEN or Ad5-Δp85 transfection on the expression of CDK2, p27, caspase-9 and -3, and poly(ADP-ribose) polymerase. H1299 cells were treated with medium alone (–) or incubated for 72 h with the indicated titers (particles/cell) of Ad5-PTEN, Ad5-Δp85, or Ad5-CMV and lysed. Lysates (30 μg/sample) were subjected to Western blot analysis. Western blot analysis for β-actin was performed to determine the relative amounts of protein loaded per well.
      Δp85 Inhibits the Activity of MAP Kinases—Δp85 induced apoptosis of NSCLC cells whereas PTEN did not. Therefore, we hypothesized that inhibition of the PI3K/AKT pathway was required but not sufficient to induce apoptosis. We sought to identify additional survival signals typically activated by peptide growth factors that are inhibited by Δp85. Receptor tyrosine kinases maintain NSCLC cell survival, in part, by activating MAP kinases (
      • Schlessinger J.
      ). We investigated the role of MAP kinases in Δp85-induced cell death. H1299 NSCLC cells were incubated with Ad5-PTEN or Ad5-Δp85, treated with EGF, and subjected to in vitro kinase assays of JNK and ERK activity (Fig. 5). ERK activity increased in cells incubated with Ad5-CMV. Ad5-PTEN and Ad5-Δp85 had similar, dose-dependent effects on ERK activity. Relative to the effect of Ad5CMV, ERK activity increased with low dose (103 particles/cell) and decreased with high dose (5 × 103 or 104 particles/cell) Ad5-PTEN or Ad5-Δp85. JNK activity decreased minimally after Ad5-PTEN and, to a much greater extent, after Ad5-Δp85 incubation. Thus, Δp85 was unique in its ability to inhibit JNK activity.
      Figure thumbnail gr5
      Fig. 5Effects of Ad5-PTEN or Ad5-Δp85 transfection on EGF-induced JNK and ERK activity. H1299 cells were incubated for 48 h with the indicated titers (particles/cell) of Ad5-Δp85, Ad5-PTEN, or Ad5-CMV or treated with medium alone (–). The cells were then serum-starved for 24 h, treated with EGF (50 ng/ml) for 15 min, and lysed. JNK and ERK were immunoprecipitated from 100-μg aliquots of the cell lysate and subjected to immune complex kinase assays (KA) using GST-c-Jun and MBP as substrates to examine the activities of JNK and ERK, respectively. As a control, JNK1 and ERK1/2 expression were examined by Western blot analysis (W) of whole cell lysates.
      We investigated the mechanism by which Δp85 inhibited JNK. p85α associates with Rac-1, an upstream activator of JNK, and activates Rac-1 through association with a multiprotein complex that binds to p85 SH2 domains (
      • Innocenti M.
      • Fritolli E.
      • Ponzanelli I.
      • Falck J.R.
      • Brachman S.M.
      • Di Fiore P.P.
      • Scita G.
      ). We investigated whether wild-type p85α and Δp85 differ in their ability to activate Rac-1. We quantitated Rac-1 activity in cell extracts using a pull-down assay with a GST-tagged PBD of PAK-1, which associates selectively with GTP-bound (activated) Rac-1 or CDC42. PBD-associated proteins are subjected to Western analysis to quantitate Rac-1 and CDC42. We performed this experiment in COS-7 cells, in which peptide growth factors activate Rac-1 through a PI3K-dependent mechanism (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ,
      • Nimnual A.S.
      • Yatsula B.A.
      • Bar-Sagi D.
      ). Using this assay we showed that Rac-1 is activated by treatment with EGF or IGF-1 (Fig. 6A). COS cells were co-transfected with wild-type p85 and increasing amounts of Δp85 and treated with EGF to activate Rac-1. Relative to the effect of wild-type p85, Δp85 inhibited peptide growth factor-induced activation of Rac-1 but not CDC42 (Fig. 6B). Thus, in contrast to the stimulatory effect of p85α, Δp85 inhibited Rac-1.
      Figure thumbnail gr6
      Fig. 6Effect of Δp85 on Rac-1 activation by peptide growth factors.A, COS-7 cells were treated for 15 min with 50 ng/ml IGF-1, EGF, or medium alone. B, COS-7 cells were co-transfected overnight with plasmids containing HA-p85 and increasing doses of Δp85, serum-starved for 16 h, and treated with IGF-1 for 15 min. Cells were lysed, and Rac-1 activity was analyzed by performing a pull-down assay on cell extracts using a GST-tagged PBD of PAK-1, followed by Western analysis (W) to quantitate PAK-1-associated Rac-1 or CDC42. To examine relative transfection efficiencies in each sample, Western analysis of HA expression was performed on total cell extracts.
      p85α serves both to stabilize p85 protein and to inactivate PI3K lipid kinase activity (
      • Cuevas B.D.
      • Lu Y.
      • Mao M.
      • Zhang J.
      • LaPushin R.
      • Siminovitch K.
      • Mills G.B.
      ). Therefore, we tested the hypothesis that Ad5-Δp85 inhibits intracellular signaling activity by increasing intracellular p85 protein levels. We incubated H1299 NSCLC cells with various doses of Ad5-Δp85 or an adenoviral vector expressing full-length p85α (Adex1CAp85α-HA) and examined their relative effects on EGF-induced phosphorylation and activation of AKT, JNK, and ERK by Western blotting and in vitro kinase assays (Fig. 7). We measured the intensity of pAKT, GST-GSK3, total AKT, and p85 bands by densitometric scanning and corrected for differences in total p85 and AKT protein levels at each virus dose. pAKT levels in cells transfected with 103, 5 × 103, and 104 particles/cell of Ad5-Δp85 were 20.6, 74.6, and 56.7%, respectively, of pAKT levels in cells transfected with the same doses of Adex1CAp85α-HA (Fig. 7, top). In vitro kinase assays using GST-GSK3 as substrate demonstrated that Ad5-Δp85 inhibited AKT kinase activity to a greater extent than Adex1CAp85α-HA (Fig. 7, top). In contrast to Ad5-Δp85, Adex1CAp85α-HA increased the phosphorylation and activity of JNK and minimally increased ERK activity (Fig. 7, middle and bottom). Treatment with LY294002 did not block the effects of Adex1CAp85α-HA on JNK and ERK (data not shown), providing evidence that PI3K activity was not required. These findings indicate that increasing the levels of wild-type p85α was not sufficient to recapitulate the effects of Δp85 on AKT, JNK, and ERK.
      Figure thumbnail gr7
      Fig. 7Effect of wild-type p85α and Δp85 on AKT, JNK, and ERK. H1299 cells were incubated with no virus (lanes designated – and EGF), empty vector (Ad5) at 104 particles/cell, Adex1CAp85α-HA (Ad-WTp85), which expresses wild-type p85α, or Ad5-Δp85 at 103, 5 × 103, or 104 particles/cell. After transfection, the cells were grown in complete medium at 37 °C for 48 h. The cells were then subjected to serum-free conditions for 12 h (–), treated for 15 min with EGF (50 ng/ml), and lysed. Lysates were subjected to either Western blot analysis (20 μg/sample) using antibodies to the indicated proteins or immunoprecipitation (100 μg/sample) to isolate AKT, JNK, and ERK for in vitro kinase assays using GST-GSK3, GST-c-Jun, and MBP, respectively, as substrates.
      MAP Kinase Signaling Contributes to NSCLC Cell Survival—We investigated the importance of JNK and ERK inhibition in Δp85-induced cell death by examining whether co-transfection of upstream activators of these kinases would block Δp85-induced cell death. In COS-7 cells, a constitutively active mutant Rac-1 (Val12) blocked Δp85-induced inhibition of JNK and ERK, whereas a constitutively active mutant MAPK/ERK kinase (MKK1) (R4F) blocked Δp85-induced inhibition of ERK but not JNK (Fig. 8A). In H1299 NSCLC cells, Ad5-Δp85-induced cell death was abrogated by Rac-1 (Val12) but not by MKK1 (R4F) (Fig. 8, B and C). Together, these findings indicate that Δp85-induced cell death requires inhibition of JNK but not ERK.
      Figure thumbnail gr8
      Fig. 8Effects of constitutively active mutants of MKK1 (R4F) and Rac-1 (Val12) on Ad5-Δp85-induced MAP kinase inhibition and apoptosis.A, COS-7 cells were transiently transfected for 6 h with 5-μg plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector using FuGENE. The cells were then transfected with Ad5-Δp85 or Ad5-CMV (1 × 103 or 5 × 103 particles/cell) and incubated for 24 h in complete medium. The cells were then changed to serum-free medium for 24 h, treated with IGF-1 (50 ng/ml) for 15 min, and lysed. JNK and ERK were immunoprecipitated from 100-μg aliquots of total cell lysates and subjected to kinase assays (KA) using GST-c-Jun and MBP, respectively, as substrates. As a control, JNK1 and ERK1/2 levels were examined by Western blot analysis (W). B, H1299 cells were transfected with 5 μg of plasmids containing Rac-1 (Val12), MKK1 (R4F), or empty vector (–). The next day, the cells were incubated with Ad5-CMV at 5 × 103 particles/cell (–) or Ad5-Δp85 at 1 × 103 or 5 × 103 particles/cell. Two days later, floating and adherent cells were isolated, fixed in 1% paraformaldehyde and 70% ethanol, stained with propidium iodide and APOBrdUrd, and subjected to flow cytometric analysis. The percentage of dead cells was determined by quantification of the pre-G1 cell population and is indicated in the upper right corner of each flow diagram. C, H1299 cells were transfected with Rac-1 (Val12) and then incubated with increasing amounts of Ad5-Δp85, as described in panel b. Genomic DNA was isolated from floating and adherent cells and subjected to 1.5% gel electrophoresis for DNA fragmentation analysis.
      On the basis of these findings, we hypothesized that Rac-1 and its downstream mediators (PAK-1/MKK4/JNK) cooperate with PI3K-dependent signaling to maintain cell survival. To test this hypothesis, we examined whether PI3K inhibition would be sufficient to induce apoptosis of MKK4-null MEF cells. These cells did not activate JNK in response to EGF (Fig. 9A). We treated MKK4-null and wild-type MEF cells with LY294002 and examined them for evidence of apoptosis. LY294002 treatment induced apoptosis in MKK4-null MEF cells, but wild-type MEF cells demonstrated minimal evidence of cell death (Fig. 9, B and C). Together, these findings support the hypothesis that PI3K- and MKK4-dependent pathways cooperate to maintain cell survival.
      Figure thumbnail gr9
      Fig. 9Relative to wild-type (+/+) MEF cells, MKK4-null (–/–) cells exhibit defects in JNK activation in response to specific stress activators and enhanced apoptosis in response to LY294002.A, MEF cells were serum-starved overnight and then subjected to no treatment (–) or treatment with UV light (60 J/m2 for 30 min), tumor necrosis factor-α (TNF) (30 ng/ml for 30 min), or EGF (50 ng/ml for 30 min). JNK was immunopurified and subjected to kinase assays using GST-c-Jun as substrate. B and C, MEF cells were treated for 3 days with the indicated doses of LY294002 or medium alone (–) and subjected to (B) DNA fragmentation assay and (C) TUNEL analysis as described under “Experimental Procedures.” The relative MKK4 expression levels in MKK4-null and wild-type MEF cells were examined by Western blot analysis. The percentages of apoptotic cells as determined by TUNEL analysis are indicated in the upper left corner of each flow diagram.

      DISCUSSION

      Cancer cells with PTEN gene loss require activation of the PI3K-dependent pathway for survival. However, PI3K-dependent signaling is activated in cancer cells through mechanisms other than PTEN gene loss, as demonstrated in NSCLC cells, which typically have evidence of PI3K pathway activation and express wild-type PTEN (
      • Brognard J.
      • Clark A.S.
      • Ni Y.
      • Dennis P.A.
      ,
      • Massion P.
      • Kuo W.L.
      • Stokoe D.
      • Olshen A.B.
      • Treseler P.A.
      • Chin K.
      • Chen C.
      • Polikoff D.
      • Jain A.N.
      • Pinkel D.
      • Albertson D.G.
      • Jablons D.M.
      • Gray J.W.
      ,
      • Forgacs E.
      • Biesterveld E.J.
      • Sekido Y.
      • Fong K.
      • Muneer S.
      • Wistuba I.I.
      • Milchgrub S.
      • Brezinschek R.
      • Virmani A.
      • Gazdar A.F.
      • Minna J.D.
      ,
      • Moore S.M.
      • Rintoul R.C.
      • Walker T.R.
      • Chilvers E.R.
      • Haslett C.
      • Sethi T.
      ,
      • Yokomizo A.
      • Tindall D.J.
      • Drabkin H.
      • Gemmill H.
      • Franklin W.A.
      • Yang P.
      • Sugio K.
      • Smith D.I.
      • Liu D.
      ,
      • Soria J.C.
      • Lee H.Y.
      • Lee J.I.
      • Wang L.
      • Issa J.P.
      • Kemp B.L.
      • Liu D.D.
      • Kurie J.M.
      • Khuri F.R.
      ). Here we investigated the role of PI3K in the survival of a NSCLC cell line that expresses wild-type PTEN. We found that LY294002 treatment and PTEN overexpression arrested cells in the G0/G1 phase of the cell cycle with minimal evidence of apoptosis. Unexpectedly, we found that Δp85 induced marked apoptosis.
      Several lines of evidence presented here support the hypothesis that Δp85 induced apoptosis through the combined inhibition of MAP kinase- and PI3K-dependent pathways. First, in addition to inhibiting AKT, Δp85 inhibited JNK activity. Second, constitutively active Rac-1 (Val12) blocked Δp85-induced apoptosis of H1299 cells. Third, LY294002 treatment induced apoptosis in MKK4-null MEF cells but not wild-type MEF cells. The cooperative effect between PI3K and MAP kinase pathways was specific to JNK-dependent signaling, as introduction of constitutively active mutant MKK1 did not block apoptosis of H1299 cells induced by Δp85. This finding is consistent with previous reports that MAP kinase family members play distinct biological roles in tumor cells (
      • Gupta S.
      • Plattner R.
      • Der C.J.
      • Stanbridge E.J.
      ,
      • Shields J.M.
      • Mehta H.
      • Pruitt K.
      • Der C.J.
      ). Together, these findings indicate that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.
      Mechanisms by which Δp85 inhibited JNK activity have not been fully defined. Introduction of wild-type p85α did not recapitulate the effects of Δp85 on JNK and AKT, suggesting that Δp85 functions through mechanisms other than increasing intracellular levels of p85α, which inactivates PI3K lipid kinase activity through changes in the stoichiometry of p85:p110 (
      • Cuevas B.D.
      • Lu Y.
      • Mao M.
      • Zhang J.
      • LaPushin R.
      • Siminovitch K.
      • Mills G.B.
      ). Alternatively, Δp85 may inhibit recruitment of p85-associated proteins required for activation of MAP kinase pathways by receptor tyrosine kinases. Supporting this possibility, we found that Δp85 inhibited Rac-1 activation by peptide growth factors. The inhibitory effect of Δp85 on peptide growth factor-induced Rac-1 activation in lung cancer cells is not consistent with previous reports that overexpression of Δp85 stimulates Rac-1 activity in T lymphocytes (
      • Kang H.
      • Schneider H.
      • Rudd C.E.
      ). Although we have yet to identify the mechanism by which Δp85 inhibits Rac-1, cell type-specific factors may be important. Recent findings indicate that p85 activates Rac-1 by associating with a multiprotein complex (including Eps8, Abi1, and SOS-1) that binds to p85 SH2 domains (
      • Innocenti M.
      • Fritolli E.
      • Ponzanelli I.
      • Falck J.R.
      • Brachman S.M.
      • Di Fiore P.P.
      • Scita G.
      ). We hypothesize that, in certain cell types, the p85 inter-SH2 domain is also required for Rac-1 activation. Recent findings have shown that this region of p85 contains several motifs that, in addition to binding to the p110 catalytic subunit, interact with other factors regulated by GTPase- and tyrosine kinase-dependent pathways (
      • Tung T.O.
      • Rodeck U.
      • Chan A.M.
      • Kimmelman A.C.
      • Rittenhouse S.E.
      • Panayotou G.
      • Tsichlis P.N.
      ), supporting the possibility that another multiprotein complex associates with this region. Additional studies will be needed to identify these proteins and to examine their role in Rac-1 activation.
      Although adenoviral vectors expressing PTEN and Δp85 shared the ability to inhibit AKT activity, they differed in other downstream signaling events. Ad5-PTEN increased p27 levels and decreased CDK2 levels, which has been described previously in cells transfected with adenoviral vectors expressing PTEN (
      • Li D.M.
      • Sun H.
      ,
      • Gottschalk A.R.
      • Basila D.
      • Wong M.
      • Dean N.M.
      • Brandts C.H.
      • Stokoe D.
      • Haas-Kogan D.A.
      ) and is consistent with the G0/G1 proliferative arrest we observed. In contrast to Ad5-PTEN, Ad5-Δp85 did not increase p27 or decrease CDK2 levels. This finding was somewhat surprising, given the ability of Δp85 to inhibit PI3K-dependent signaling. Analysis of the dose-dependent effects of Ad5-PTEN and Ad5-Δp85 demonstrated that Ad5-PTEN was a more potent inhibitor of pAKT levels than was Ad5-Δp85, which could account for their differences in downstream signaling. The adenoviral dose-dependent changes in ERK activity we observed in NSCLC cells transfected with Ad5-PTEN differs from observations in glioblastoma and prostate cancer cells transfected with Ad-PTEN and in PTEN-null embryonic stem cells, in which ERK activity did not change (
      • Lu Y.
      • Lin Y.Z.
      • LaPushin R.
      • Cuevas B.
      • Fang X.
      • Yu S.X.
      • Davies M.A.
      • Khan H.
      • Furui T.
      • Mao M.
      • Zinner R.
      • Hung M.C.
      • Steck P.
      • Siminovitch K.
      • Mills G.B.
      ,
      • Davies M.A.
      • Koul D.
      • Dhesi H.
      • Berman R.
      • McDonnell T.J.
      • McConkey D.
      • Yung W.K.A.
      • Steck P.
      ,
      • Sun H.
      • Lesche R.
      • Li D.M.
      • Liliental J.
      • Zhang H.
      • Gao J.
      • Gavrilova N.
      • Mueller B.
      • Liu X.
      • Wu H.
      ,
      • Wu X.
      • Senechal K.
      • Neshat M.S.
      • Whang Y.E.
      • Sawyers C.L.
      ). This difference could be the result of cell type-specific factors or nonspecific effects of exogenous PTEN in H1299 cells.
      Previous studies have shown that MKK4 expression and activity are altered in human tumor cells and that MKK4 can act as both a promoter and a suppressor of human tumorigenesis. The MKK4 gene is deleted or mutated in a subgroup of pancreatic, biliary, and breast carcinomas, and reintroduction of MKK4 inhibits the metastatic ability of certain tumor cells, demonstrating that MKK4 has tumor suppressor activity (
      • Teng D.H.F.
      • Perry 3rd, W.L.
      • Hogan J.K.
      • Baumgard M.
      • Bell R.
      • Berry S.
      • Davis T.
      • Frank D.
      • Frye C.
      • Hattier T.
      • Hu R.
      • Jammulapati S.
      • Janecki T.
      • Leavitt A.
      • Mitchell J.T.
      • Pero R.
      • Sexton D.
      • Schroeder M.
      • Su P.H.
      • Swedlund B.
      • Kyriakis J.M.
      • Avruch J.
      • Bartel J.
      • Wong A.K.
      • Tavtigian S.V.
      ,
      • Yoshida B.A.
      • Dubauskas Z.
      • Chekmareva M.A.
      • Christiano T.R.
      • Stadler W.M.
      • Rinker-Schaeffer C.W.
      ,
      • Su G.
      • Hilgers W.
      • Shekher M.C.
      • Tang D.J.
      • Yeo C.J.
      • Hruban R.H.
      • Kern S.E.
      ). Potentially mediating this effect, Ras pathway activation increases the expression of p53 and p16INK4a, which induces premature cellular senescence; conversely, inactivation of p53 or p16 prevents Ras-induced growth arrest (
      • Serrano M.
      • Lin A.W.
      • McCurrach M.E.
      • Beach D.
      • Lowe S.W.
      ). In contrast to these studies, MKK4 is known to be a downstream mediator of Rac-1, and Rac-1 activation contributes to Ras-induced cellular transformation (
      • Qiu R.G.
      • Chen J.
      • Kirn D.
      • McCormick F.
      • Symons F.
      ), indicating that MKK4 plays a role in cellular transformation. Supporting the latter hypothesis, we found that Rac-1 activation rescued lung cancer cells from Δp85-induced apoptosis, and MKK4 cooperated with PI3K to maintain MEF cell survival. This finding supports in vitro studies of lung cancer cells demonstrating that MKK4-dependent pathways play a dominant role in mutant Ras-induced colony formation (
      • Bost F.
      • McKay R.
      • Bost M.
      • Potapova O.
      • Dean N.M.
      • Mercola D.
      ,
      • Xiao L.
      • Lang W.
      ). Thus, MKK4 and its downstream mediators play apparently contradictory roles in the regulation of cellular growth and transformation that may depend on the presence of cell type-specific factors or the activity of tumor suppressor pathways that inhibit the mitogenic and transforming effects of MKK4.
      Findings presented here have implications for the design of effective therapeutic approaches for lung cancer. Signal transduction inhibitors are being assessed in clinical trials as therapeutic agents for several types of cancer. The enthusiasm for these agents has been fueled by the efficacy of ABL kinase inhibitors in the treatment of chronic myelogenous leukemia, which arises from a reciprocal chromosomal translocation involving the Bcr and Abl genes (
      • Druker B.
      • Talpaz J.
      • Resta M.
      • Peng D.J.
      • Buchdunger B.
      • Ford E.
      • Lydon J.M.
      • Kantarjian N.B.
      • Capdeville H.
      • Ohno-Jones R.
      • Sawyers C.L.
      ). However, unlike chronic myelogenous leukemia, in which constitutively active Abl is sufficient to induce the disease (
      • Honda H.
      • Oda H.
      • Suzuki T.
      • Takahashi T.
      • Witte O.
      • Ozawa K.
      • Ishikawa T.
      • Yazaki Y.
      • Harai H.
      ), lung tumorigenesis is a multistep process leading to aberrant activity of a variety of oncogenic and tumor suppressive pathways. These pathways act in combination to induce malignant transformation of NHBE cells and to maintain the survival of lung cancer cells (
      • Fong K.M.
      • Sekido Y.
      • Minna J.D.
      ). Thus, in patients with lung cancer, inhibition of multiple pathways may be necessary to induce tumor regression. The findings presented here support the hypothesis that PI3K- and MKK4/JNK-dependent pathways cooperate in lung cancer cells to maintain their survival, and combination therapy targeting these pathways should be considered in future clinical trials.

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