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Phosphatidylinositol 3-Kinase/Akt Activity Regulates c-FLIP Expression in Tumor Cells*

Open AccessPublished:March 09, 2001DOI:https://doi.org/10.1074/jbc.C000569200
      The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.
      TNF
      tumor necrosis factor
      DD
      death domain
      FADD
      Fas-associated death domain
      FLICE
      FADD-homologous ICE-like protease
      FLIP
      FLICE-inhibitory protein
      ERK
      extracellular signal-regulated kinase
      pERK
      phosphorylated ERK
      p-Akt
      phosphorylated Akt
      GSK3a/b
      glycogen synthase kinase 3a/b
      HA
      hemagglutinin
      MAP
      mitogen-activated protein
      MEK
      MAP/ERK kinase
      PI
      phosphatidylinositol
      CREB
      cAMP-response element-binding protein
      RT-PCR
      reverse transcriptase-polymerase chain reaction
      FCS
      fetal calf serum
      DN-Akt
      dominant negative Akt adenoviral construct
      cIAP
      cellular inhibitor of apoptosis protein
      TRAIL
      TNF-related apoptosis-inducing ligand
      The p55 TNF1 receptor, Fas, LARD (DR3), and the TRAIL receptors DR4 and DR5 are members of the TNF receptor family that contain a unique cytoplasmic sequence termed a death domain (DD). Upon engagement by ligand or antibody cross-linking, these receptors trimerize and recruit an adapter protein called FADD (Fas-associated deathdomain) (
      • Chinnaiyan A.M.
      • Tepper C.G.
      • Seldin M.F.
      • O'Rourke K.
      • Kischkel F.C.
      • Hellbardt S.
      • Krammer P.H.
      • Peter M.E.
      • Dixit V.M.
      ,
      • Boldin M.P.
      • Goncharov T.M.
      • Goltsev Y.V.
      • Wallach D.
      ,
      • Chinnaiyan A.M.
      • O'Rourke K.
      • Tewari M.
      • Dixit V.M.
      ), which interacts through its own DD with those of the receptors or other adapters (e.g. TRADD). The resulting multimolecular complex, referred to as a death-inducing signaling complex or DISC, recruits an inactive procaspase called FLICE or caspase-8, which is then cleaved to an active form, initiating a cascade of proteolytic events resulting in the cleavage of numerous proteins essential for DNA repair and the maintenance of cell viability (
      • Kataoka T.
      • Schroter M.
      • Hahne M.
      • Schneider P.
      • Irmler M.
      • Thome M.
      • Froelich C.J.
      • Tschopp J.
      ). It has been shown that FADD multimerization is sufficient to kill some cells even in the absence of TNF family ligands or receptor cross-linking (
      • Micheau O.
      • Solary E.
      • Hammann A.
      • Dimanche-Boitrel M.-T.
      ). For example, the apoptosis induced by certain chemotherapeutic agents is thought to rely on ligand-independent FADD multimerization, since it can be blocked by a dominant negative FADD (
      • Micheau O.
      • Solary E.
      • Hammann A.
      • Dimanche-Boitrel M.-T.
      ). Collectively, these data suggest that FADD plays a critical role in apoptosis induced by the cross-linking of DD-containing membrane receptors of the TNF receptor family (e.g. Fas) as well as that resulting from exposure to cytotoxic drugs.
      Many tumors express Fas (
      • Houghton J.A.
      • Harwood F.G.
      • Gibson A.A.
      • Tillman D.M.
      ) and TRAIL receptors (
      • Zhang X.D.
      • Franco A.
      • Myers K.
      • Gray C.
      • Nguyen T.
      • Hersey P.
      ,
      • Zhang X.D.
      • Franco A.V.
      • Nguyen T.
      • Gray C.P.
      • Hersey P.
      ) yet are resistant to apoptosis induced by exposure to Fas ligand, TRAIL, or agonist anti-receptor antibodies. It has been proposed that this resistance is due to the presence of one or more inhibitors of apoptosis (
      • Rothstein T.L.
      • Wang J.K.
      • Panka D.J.
      • Foote L.C.
      • Wang Z.
      • Stanger B.
      • Cui H.
      • Ju S.T.
      • Marshak-Rothstein A.
      ). Numerous cellular proteins, including FLIP, Fas-associated protein (FAP-1) (
      • Sato T.
      • Irie S.
      • Kitada S.
      • Reed J.C.
      ), Bcl-2, Bcl-X, Bruton's tyrosine kinase (
      • Vassilev A.
      • Ozer Z.
      • Navara C.
      • Mahajan S.
      • Uckun F.M.
      ), Toso (
      • Hitoshi Y.
      • Lorens J.
      • Kitada S.I.
      • Fisher J.
      • LaBarge M.
      • Ring H.Z.
      • Francke U.
      • Reed J.C.
      • Kinoshita S.
      • Nolan G.P.
      ), and SODD (silencerof death domain) (
      • Tschopp J.
      • Martinon F.
      • Hofmann K.
      ,
      • Jiang Y.
      • Woronicz J.D.
      • Liu W.
      • Goeddel D.V.
      ) as well as caspase inhibitors such as cIAP (
      • Takahashi R.
      • Deveraux Q.
      • Tamm I.
      • Welsh K.
      • Assa-Munt N.
      • Salvesen G.S.
      • Reed J.C.
      ) and survivin (
      • Tamm I.
      • Wang Y.
      • Sausville E.
      • Scudiero D.A.
      • Vigna N.
      • Oltersdorf T.
      • Reed J.C.
      ,
      • Jaattela M.
      ), have been shown to inhibit the death process, either early on at the level of Fas (FAP-1) or further downstream. FLIP is a cytoplasmic protein that has sequence homology to FLICE. FLIP is capable of binding to FADD yet is unable to be cleaved to an active caspase because of a substitution of a tyrosine for an active site cysteine, thus preventing the initiation of the death pathway (
      • Irmler M.
      • Thome M.
      • Hahne M.
      • Schneider P.
      • Hofmann K.
      • Steiner V.
      • Bodmer J.L.
      • Schroter M.
      • Burns K.
      • Mattmann C.
      • Rimoldi D.
      • French L.E.
      • Tschopp J.
      ,
      • Hu S.
      • Vincenz C.
      • Ni J.
      • Gentz R.
      • Dixit V.M.
      ,
      • Rasper D.M.
      • Vaillancourt J.P.
      • Hadano S.
      • Houtzager V.M.
      • Seiden I.
      • Keen S.L.
      • Tawa P.
      • Xanthoudakis S.
      • Nasir J.
      • Martindale D.
      • Koop B.F.
      • Peterson E.P.
      • Thornberry N.A.
      • Huang J.
      • MacPherson D.P.
      • Black S.C.
      • Hornung F.
      • Lenardo M.J.
      • Hayden M.R.
      • Roy S.
      • Nicholson D.W.
      ). FLIP has been demonstrated to play a role in the prevention of apoptosis in a number of systems, especially those involving the immune system. Cells with high levels of FLIP relative to FLICE are generally resistant to apoptosis (
      • Scaffidi C.
      • Schmitz I.
      • Krammer P.H.
      • Peter M.E.
      ), whereas cells with low levels of FLIP relative to FLICE are more sensitive to apoptosis (
      • Scaffidi C.
      • Schmitz I.
      • Krammer P.H.
      • Peter M.E.
      ). When naive T cells are initially activated, they express Fas yet are resistant to Fas-mediated apoptosis (
      • Ettinger R.
      • Panka D.J.
      • Wang J.
      • Stanger B.
      • Ju S.-T.
      • Marshak-Rothstein A.
      ,
      • Refaeli Y.
      • Parijs L.V.
      • London C.A.
      • Tschopp J.
      • Abbas A.K.
      ). However, when these T cells are rechallenged with antigen, they become sensitive to Fas-mediated apoptosis, even though the expression of Fas on the surface of these cells is unchanged. This change in susceptibility to Fas-mediated apoptosis correlated with FLIP levels, which are high during the initial stimulation but significantly reduced upon rechallenge (
      • Boldin M.P.
      • Goncharov T.M.
      • Goltsev Y.V.
      • Wallach D.
      ,
      • Algeciras-Schimnich A.
      • Griffith T.S.
      • Lynch D.H.
      • Paya C.V.
      ,
      • Kataoka T.
      • Schroter M.
      • Hahne M.
      • Schneider P.
      • Irmler M.
      • Thome M.
      • Froelich C.J.
      • Tschopp J.
      ). Interleukin-2 has been shown to markedly enhance the susceptibility of activated T cells to Fas-mediated apoptosis and at the same time down-modulate FLIP (
      • Refaeli Y.
      • Parijs L.V.
      • London C.A.
      • Tschopp J.
      • Abbas A.K.
      ), the loss of which may account for the interleukin-2 effect. Recently, the protection from Fas-mediated apoptosis in B cells upon cross-linking of the B cell receptor has been shown to be associated with an increase in FLIP expression (
      • Wang J.
      • Lobito A.A.
      • Shen F.
      • Hornung F.
      • Winoto A.
      • Lenardo M.J.
      ). Thus, at least for B and T lymphocytes, susceptibility to apoptosis induced by death receptor ligation is governed in part by the level of FLIP.
      In addition to the ability of FLIP to act as a competitive inhibitor of the binding of caspase-8 to FADD, it has also been shown to induce the activation of NF-κB via TNF receptor-1 through TRADD, TRAF-2, RIP, NIK, and IKK (
      • Hu W-H.
      • Johnson H.
      • Shu H.-B.
      ), thus providing a second anti-apoptotic mechanism for FLIP.
      Although initially described as a viral product (
      • Thome M.
      • Schneider P.
      • Hofman K.
      • Fickenscher H.
      • Meinl E.
      • Neipel F.
      • Mattmann C.
      • Burns K.
      • Bodmer J.L.
      • Schroter M.
      • Scaffidi C.
      • Krammer P.H.
      • Peter M.E.
      • Tschopp J.
      ), a cellular analogue of FLIP has since been discovered, and high levels are commonly found in tumors (
      • Irmler M.
      • Thome M.
      • Hahne M.
      • Schneider P.
      • Hofmann K.
      • Steiner V.
      • Bodmer J.L.
      • Schroter M.
      • Burns K.
      • Mattmann C.
      • Rimoldi D.
      • French L.E.
      • Tschopp J.
      ,
      • Tschopp J.
      • Irmler M.
      • Thome M.
      ). In one study in melanoma cells, the levels of FLIP were found to correlate inversely with susceptibility to apoptosis induced by exposure to TRAIL (
      • Griffith T.S.
      • Chin W.A.
      • Jackson G.C.
      • Lynch D.H.
      • Kubin M.Z.
      ). However, other studies have failed to identify a linkage between FLIP expression and sensitivity to Fas or TRAIL (
      • Zhang X.D.
      • Franco A.
      • Myers K.
      • Gray C.
      • Nguyen T.
      • Hersey P.
      ).
      Very little is known about the signaling pathways or transcription elements that control the expression of FLIP. In activated T cells, FLIP expression has been shown to be dependent on the ERK/MAP kinase pathway because the addition of a dominant active MKK1 (MEK-1) induces FLIP in 293T and Jurkat cells (
      • Yeh J.-H.
      • Hsu S.-C.
      • Han S.-H.
      • Lai M.-Z.
      ). The molecular basis for the high constitutive levels of FLIP observed in many tumor cell lines is unknown.
      PI 3-kinase has been shown to protect cells from apoptosis in a caspase-dependent manner (
      • Berra E.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Chen R.H.
      • Su Y.H.
      • Chuang R.L.
      • Chang T.Y.
      ,
      • Gibbs B.F.
      • Grabbe J.
      ). PI 3-kinase catalyzes the phosphorylation of phosphoinositol-4 phosphate and phosphoinositol-4,5 phosphosphate at the 3 position. Kinases such as PDK1 and pkB/Akt bind to these phosphorylated intermediates via their pleckstrin homology domain. PDK1 in turn phosphorylates and activates pkB/Akt (
      • Duronio V.
      • Scheid M.P.
      • Ettinger S.
      ), which then phosphorylates several proteins that have been implicated in the control of cell survival (
      • Berra E.
      • Diaz-Meco M.T.
      • Moscat J.
      ,
      • Gibbs B.F.
      • Grabbe J.
      ,
      • Wennstrom S.
      • Downward J.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • Capodici C.
      • Hanft S.
      • Feoktistov M.
      • Pillinger M.H.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). The phosphorylation of procaspase-9 by Akt, for example, inactivates this protease, blocking the propagation of death signals originating in the mitochondria (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). Likewise, Akt-mediated phosphorylation of Bad results in the binding of this pro-apoptotic Bcl-2 family member to 14-3-3 proteins and its dissociation from the mitochondria (
      • Zha J.
      • Harada H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ,
      • Zha J.
      • Harada H.
      • Osipov K.
      • Jockel J.
      • Waksman G.
      • Korsmeyer S.J.
      ,
      • Hsu S.Y.
      • Kaipia A.
      • Zhu L.
      • Hsueh A.J.
      ). Some targets of pkB/Akt, such as NF-κB (
      • Kane L.P.
      • Shapiro V.S.
      • Stokoe D.
      • Weiss A.
      ), CREB (
      • Du K.
      • Montminy M.
      ,
      • Pugazhenthi S.
      • Nesterova A.
      • Sable C.
      • Heidenreich K.A.
      • Boxer L.M.
      • Heasley L.E.
      • Reusch J.E.
      ), and Forkhead (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ), are transcription factors that regulate cell survival. In some cells, the anti-apoptotic effect of NF-κB activation is partly attributable to the expression of cIAP, a potent caspase inhibitor (
      • Wang C.Y.
      • Mayo M.W.
      • Korneluk R.G.
      • Goeddel D.V.
      • Baldwin A.S.
      ). The phosphorylation of Forkhead by Akt blocks its activity, reducing the levels of expression of pro-apoptotic proteins such as Fas ligand (
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P.
      • Hu L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). These data illustrate the diversity of downstream effectors through which the PI 3-kinase/Akt signaling pathway affects cell survival.
      The ability of a constitutively active MEK1 to induce FLIP expression in T cell lines and the multiplicity of anti-apoptotic proteins in which expression and/or function are regulated through the PI 3-kinase pathway suggest that the constitutive expression of FLIP by tumor cells might be attributable to the activation of either the Ras-Raf-MEK-ERK and/or PI 3-kinase pathways. To test these hypotheses, we carried out a series of studies with drugs and a dominant negative kinase that inhibit these pathways. The results of our studies clearly implicate the PI 3-kinase/Akt pathway as the predominant regulator of FLIP expression in tumor cells.

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