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Substrate Specificity of αvβ3Integrin-mediated Cell Migration and Phosphatidylinositol 3-Kinase/AKT Pathway Activation*

  • Duo-Qi Zheng
    Affiliations
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Amy S. Woodard
    Footnotes
    Affiliations
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Giovanni Tallini
    Affiliations
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Lucia R. Languino
    Footnotes
    Affiliations
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants CA-71870 and DK-52670 and Army Prostate Cancer Research Program Grant DAMD17-98-1-8506 (to L. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Present address: Genetics Institute, One Burtt Rd., Andover, MA 01810.
    § To whom correspondence and requests for reprints should be addressed: Dept. of Pathology, Yale University School of Medicine, P. O. Box 208023, 310 Cedar St., New Haven, CT 06520. Tel.: 203-737-1454; Fax: 203-737-1455; E-mail: [email protected]
Open AccessPublished:August 11, 2000DOI:https://doi.org/10.1074/jbc.M002646200
      The αvβ3integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express αvβ3adhere and migrate on osteopontin and vitronectin in an αvβ3-dependent manner. However, exogenous expression of αvβ3 in noninvasive prostate cancer LNCaP (β3-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of αvβ3 by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that αvβ3-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, αvβ3 engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of β3-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in β3-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, αvβ3mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in β3-LNCaP cells, αvβ3 mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support αvβ3-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that αvβ3 exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.
      VN
      vitronectin
      OPN
      osteopontin
      FN
      fibronectin
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PAGE
      polyacrylamide gel electrophoresis
      BSA
      bovine serum albumin
      FACS
      fluorescence-activated cell sorter
      FAK
      focal adhesion kinase
      ERK
      extracellular signal-regulated kinase
      MAP
      mitogen-activated protein
      EGF
      epidermal growth factor
      HER
      human EGF receptor
      PBS
      phosphate-buffered saline
      Integrins are heterodimeric cell surface receptors that consist of noncovalently associated α and β subunits; these receptors have been shown to play a role in cell migration, proliferation, and gene transcription and can affect cancer cell invasion and growth (
      • Haas T.A.
      • Plow E.F.
      ,
      • Juliano R.
      ,
      • Hynes R.O.
      ). The role of αvβ3 integrin in mediating cell migration and survival has been described (
      • Eliceiri B.P.
      • Cheresh D.A.
      ,
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ,
      • Petitclerc E.
      • Stromblad S.
      • von Schalscha T.L.
      • Mitjans F.
      • Piulats J.
      • Montgomery A.M.
      • Cheresh D.A.
      • Brooks P.C.
      ). Exogenous expression of αvβ3 has been shown to increase melanoma tumor growth and metastases (
      • Felding-Habermann B.
      • Mueller B.M.
      • Romerdahl C.A.
      • Cheresh D.A.
      ,
      • Filardo E.J.
      • Brooks P.C.
      • Deming S.L.
      • Damsky C.
      • Cheresh D.A.
      ), to induce conversion from radical to vertical growth phase in primary human melanoma cells, and to promote melanoma cell survival in vivo (
      • Hsu M.Y.
      • Shih D.T.
      • Meier F.E.
      • Van Belle P.
      • Hsu J.Y.
      • Elder D.E.
      • Buck C.A.
      • Herlyn M.
      ) and in three-dimensional collagen gels (
      • Montgomery A.M.
      • Reisfeld R.A.
      • Cheresh D.A.
      ) indicating αvβ3 contribution at the level of motility and proliferation in vivo. We have previously shown that highly invasive and metastatic human PC3 prostate cancer cells express αvβ3 whereas nontumorigenic and noninvasive LNCaP cells do not (
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ).
      The αvβ3 integrin is a promiscuous receptor that mediates adhesion of several cell types to different ligands and of cancer cells to platelets (
      • Felding-Habermann B.
      • Cheresh D.A.
      ,
      • Felding-Habermann B.
      • Habermann R.
      • Saldivar E.
      • Ruggeri Z.M.
      ). Among others, vitronectin (VN)1- (
      • Felding-Habermann B.
      • Cheresh D.A.
      ,
      • Woodard A.S.
      • Garcı́a-Cardeña G.
      • Leong M.
      • Madri J.A.
      • Sessa W.C.
      • Languino L.R.
      ,
      • Seftor R.E.B.
      • Seftor E.A.
      • Gehlsen K.R.
      • Stetler-Stevenson W.G.
      • Brown P.D.
      • Ruoslahti E.
      • Hendrix M.J.C.
      ) and osteopontin (OPN)-coated (
      • Senger D.R.
      • Ledbetter S.R.
      • Claffey K.P.
      • Papadopoulos-Sergiou A.
      • Perruzzi C.A.
      • Detmar M.
      ,
      • Senger D.
      • Perruzzi C.A.
      ,
      • Ross F.P.
      • Chappel J.
      • Alvarez J.I.
      • Sander D.
      • Butler W.T.
      • Farach-Carson M.C.
      • Mintz K.A.
      • Robey P.G.
      • Teitelbaum S.L.
      • Cheresh D.A.
      ,
      • Hu D.D.
      • Hoyer K.R.
      • Smith J.W.
      ,
      • Miyauchi A.
      • Alvarez J.
      • Greenfield E.M.
      • Teti A.
      • Grano M.
      • Colucci S.
      • Zambonin-Zallone A.
      • Ross F.P.
      • Teitelbaum S.L.
      • Cheresh D.
      • Hruska K.A.
      ,
      • Faccio R.
      • Grano M.
      • Colucci S.
      • Zallone A.Z.
      • Quaranta V.
      • Pelletier A.J.
      ) substrates have been shown to support cell adhesion via αvβ3.
      OPN is expressed in mature bone where prostate cancer cells preferentially metastasize. A causal role for OPN during tumor progression has been suggested by several studies, including the observation that high levels of OPN support a tumorigenic and metastatic phenotype (
      • Crawford H.C.
      • Matrisian L.M.
      • Liaw L.
      ,
      • Thalmann G.N.
      • Sikes R.A.
      • Devoll R.E.
      • Kiefer J.A.
      • Markwalder R.
      • Klima I.
      • Farach-Carson C.M.
      • Studer U.E.
      • Chung L.W.K.
      ). OPN is up-regulated in prostate cancer and other carcinomas (
      • Senger D.R.
      • Asch B.B.
      • Smith B.D.
      • Perruzzi C.A.
      • Dvorak H.F.
      ,
      • Brown L.F.
      • Papadopoulos-Sergiou A.
      • Berse B.
      • Manseau E.J.
      • Tognazzi K.
      • Perruzzi C.A.
      • Dvorak H.F.
      • Senger D.R.
      ) and increases anchorage-independent growth of prostate cancer cells (
      • Thalmann G.N.
      • Sikes R.A.
      • Devoll R.E.
      • Kiefer J.A.
      • Markwalder R.
      • Klima I.
      • Farach-Carson C.M.
      • Studer U.E.
      • Chung L.W.K.
      ) as well as proliferation of normal prostate cells (
      • Elgavish A.
      • Prince C.
      • Chang P.L.
      • Lloyd K.
      • Lindsey R.
      • Reed R.
      ). Furthermore, it is found in plasma of patients with metastatic diseases, and it increases metastatic ability of transformed cells (
      • Oates A.J.
      • Barraclough R.
      • Rudland P.S.
      ,
      • Rittling S.R.
      • Denhardt D.T.
      ). Its interaction with different surface receptors has been shown: specifically, α4β1, α8β1, α9β1, αvβ1, αvβ5, and CD44 on different cell types (
      • Bayless K.J.
      • Meininger G.A.
      • Scholtz J.M.
      • Davis G.E.
      ,
      • Denda S.
      • Reichardt L.F.
      • Muller U.
      ,
      • Saegusa Y.
      • Ziff M.
      • Welkovish L.
      • Cavender D.
      ,
      • Smith L.L.
      • Giachelli C.M.
      ,
      • Haapasalmi K.
      • Makela M.
      • Oksala O.
      • Heino J.
      • Yamada K.M.
      • Uitto V.-J.
      • Larjava H.
      ,
      • Hu D.D.
      • Lin E.C.
      • Kovach N.L.
      • Hoyer J.R.
      • Smith J.W.
      ,
      • Katagiri Y.U.
      • Sleeman J.
      • Fujii H.
      • Herrlich P.
      • Hotta H.
      • Tanaka K.
      • Chikuma S.
      • Yagita H.
      • Okumura K.
      • Murakami M.
      • Saiki I.
      • Chambers A.F.
      • Uede T.
      ,
      • Liaw L.
      • Skinner M.P.
      • Raines E.W.
      • Ross R.
      • Cheresh D.A.
      • Schwartz S.M.
      • Giachelli C.M.
      ). The ability of OPN to support haptotaxis of different cell types via αvβ3 has been shown (
      • Senger D.
      • Perruzzi C.A.
      ). However, the signaling mechanisms activated via OPN-αvβ3 interaction that support cell migration have never been described.
      It has recently been shown that αvβ3 can be activated (
      • Pelletier A.J.
      • Kunicki T.
      • Ruggeri Z.M.
      • Quaranta V.
      ,
      • Shattil S.J.
      • Ginsberg M.H.
      ,
      • Byzova T.V.
      • Plow E.F.
      ) in a cell-type specific (
      • Pampori N.
      • Hato T.
      • Stupack D.G.
      • Aidoudi S.
      • Cheresh D.A.
      • Nemerow G.R.
      • Shattil S.J.
      ) manner. Its activation appears to be a sophisticated mechanism to induce adhesion to αvβ3 ligands, specifically to prothrombin via protein kinase C activation or ADP stimulation, to VN via either hepatocyte growth factor or AP5, an antibody to αvβ3, and to OPN via either AP5 or agonists, including ADP (
      • Byzova T.V.
      • Plow E.F.
      ,
      • Trusolino L.
      • Serini G.
      • Cecchini G.
      • Besati C.
      • Ambesi-Impiombato F.S.
      • Marchisio P.C.
      • De Filippi R.
      ,
      • Pelletier A.
      • Kunicki T.
      • Quaranta V.
      ,
      • Bennett J.S.
      • Chan C.
      • Vilaire G.
      • Mousa S.A.
      • DeGrado W.F.
      ). It has been recently shown that activated αvβ3 mediates cell adhesion and migration to bone sialoprotein (
      • Byzova T.V.
      • Kim W.
      • Miduar R.J.
      • Plow E.F.
      ). In one instance, upon activation by AP5, αvβ3 was shown to increase adhesion and migration of αvβ3-expressing melanoma cells on OPN and VN in a comparable manner (
      • Pelletier A.
      • Kunicki T.
      • Quaranta V.
      ). However, the role of activation-dependent and activation-independent ligands of αvβ3 in modulating cell functions and downstream signaling events has not been described.
      Several signaling molecules, specifically FAK, PI 3-kinase, and members of the MAP kinase family, play a role in modulating integrin-mediated cell migration (
      • Howe A.
      • Aplin A.E.
      • Alahari S.K.
      • Juliano R.L.
      ). FAK is a non-receptor tyrosine kinase localized in focal contacts that becomes tyrosine-phosphorylated and subsequently activated upon integrin-mediated cell adhesion to several matrix proteins, including VN (
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ,
      • Otey C.A.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      ). FAK phosphorylation of tyrosine 397 (Tyr397) is crucial for cell migration (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ). PI 3-kinase is a lipid kinase involved in proliferation, survival, and migration in response to growth factors including EGF and integrin signaling (
      • Rameh L.E.
      • Cantley L.C.
      ,
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). PI 3-kinase forms a complex with FAK via FAK-Tyr397 in response to cell adhesion or platelet-derived growth factor stimulation (
      • Chen H.-C.
      • Guan J.-L.
      ,
      • Chen H.-C.
      • Guan J.-L.
      ), and it is known to act as a downstream effector of FAK and to control FAK-induced cell migration activated by cell adhesion to extracellular matrix proteins (
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ,
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ). AKT plays an important role in transducing survival signals in response to several growth factors and β1 integrin engagement (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Downward J.
      ) and very recently in supporting vascular endothelial growth factor-induced chemotaxis (
      • Morales-Ruiz M.
      • Fulton D.
      • Sowa G.
      • Languino L.R.
      • Fujio Y.
      • Walsh K.
      • Sessa W.C.
      ). In response to integrin engagement, AKT activation is PI 3-kinase-dependent because wortmannin completely prevents AKT serine phosphorylation (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ) and is also controlled by Cdc42, a member of the GTPase family (
      • Clark E.A.
      • King W.G.
      • Brugge J.S.
      • Symons M.
      • Hynes R.O.
      ) or by ILK (
      • Delcommenne M.
      • Tan C.
      • Gray V.
      • Rue L.
      • Woodgett J.
      • Dedhar S.
      ). Integrin engagement has also been shown to stimulate activation of two members of the MAP kinase family, extracellular signal-regulated kinase-1 and -2 (ERK1/2) (
      • Juliano R.
      ), which contribute to integrin-mediated cell migration (
      • Wei J.
      • Shaw L.M.
      • Mercurio A.M.
      ,
      • Eliceiri B.P.
      • Klemke R.
      • Stromblad S.
      • Cheresh D.A.
      ).
      In this study, we show that adhesion of invasive prostate cancer PC3 cells to OPN and VN activates the PI 3-kinase/AKT signaling pathway; however, exogenous expression of αvβ3 in noninvasive LNCaP cells mediates VN binding but requires EGF stimulation to mediate binding to OPN. In these cells, adhesion to OPN does not support cell migration and PI 3-kinase/AKT pathway activation, whereas αvβ3 mediates cell migration and PI 3-kinase/AKT pathway activation on VN. These results show that αvβ3 is expressed in multiple functionally different states and is able to mediate cell migration in a substrate-specific and functional state-dependent manner; finally, they show that αvβ3 activates intracellular signaling pathways in a selective manner in response to individual ligands.

      DISCUSSION

      This study shows that αvβ3 is expressed in multiple functional states and that its ability to mediate cell migration and intracellular signaling pathways is substrate-specific and functional state-dependent. In PC3 cells, αvβ3 mediates cell adhesion, migration, and PI 3-kinase/AKT pathway activation on VN and OPN. In contrast, adhesion to OPN of noninvasive LNCaP cells upon exogenous expression of αvβ3 requires its activation by EGF although αvβ3 is in a functional state that allows adhesion to a different ligand, VN, in the absence of EGF. Furthermore, in LNCaP cells, while αvβ3 mediates cell migration and PI 3-kinase/AKT pathway activation on VN, adhesion to OPN fails to support cell migration and PI 3-kinase/AKT pathway activation mediated by αvβ3. This is the first report that shows an integrin ligand-mediated phenotypical alteration that reverts a migratory cell into a nonmigratory cell via engagement of the same integrin. We conclude that αvβ3 exists in multiple functional states that can bind either VN selectively or both VN and OPN and that can differentially activate cell migration and the PI 3-kinase/AKT signaling pathway in a ligand-specific manner. The results highlight a versatile role for αvβ3in the regulation of the PI 3-kinase/AKT pathway and in a substrate-dependent control of cell invasion.
      We show for the first time that EGF reverts a form of αvβ3 that does not recognize OPN in an OPN-binding form. Although the mechanism of activation remains to be identified, EGF effect is not due to a change in integrin expression on the cell surface because EGF regulates cell adhesion to OPN without a significant change in integrin expression (Fig. 1). EGF- downstream players that might potentially activate αvβ3 are protein kinase C, known to be involved in mediating αvβ3 activation (
      • Byzova T.V.
      • Plow E.F.
      ), and HER through its direct association with integrins (
      • Moro L.
      • Venturino M.
      • Bozzo C.
      • Silengo L.
      • Altruda F.
      • Beguinot L.
      • Tarone G.
      • Defilippi P.
      ); however, additional modulators, such as integrin-associated proteins (
      • Lindberg F.P.
      • Gresham H.D.
      • Reinhold M.I.
      • Brown E.J.
      ,
      • Hemler M.E.
      ), might be responsible for changes in ligand binding or post-ligand binding activities. Similar to our findings, activation-independent (fibrinogen) and -dependent (prothrombin) ligands for αvβ3 have been shown by Byzova and Plow (
      • Byzova T.V.
      • Plow E.F.
      ) suggesting that a sophisticated mechanism of tight regulation and ligand selection involves αvβ3.
      The EGF receptor has been shown to synergize with αvβ5 to increase cell migration (
      • Klemke R.L.
      • Yebra M.
      • Bayna E.M.
      • Cheresh D.A.
      ); LNCaP cells express low levels of β5 and large amounts of β1 (Fig. 1 and Ref.
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ). However, these previously described OPN receptors did not play a role in the adhesion of these cells to OPN in our experimental system, since first, parental or mock-LNCaP cells that did not express αvβ3did not adhere to OPN in response to EGF stimulation and second, an antibody to αvβ5 did not inhibit OPN adhesion of β3-LNCaP cells. We conclude that EGF and its receptor HER synergize with αvβ3 in a substrate-specific manner on OPN but not on VN. This change required for β3-LNCaP cell adhesion to OPN did not support cell migration on OPN although these cells migrated on VN. The ability of αvβ3 to mediate cell migration is therefore substrate-specific. Invasive PC3 cells have the ability to up-regulate cell migration through αvβ3 on OPN; therefore, it is conceivable that when cancer cells lose the ability to select their cell binding partners by uncoupling/deregulating the synergistic activity of αvβ3 integrin and HER, such as in PC3 cells, they migrate in response to engagement by multiple αvβ3 ligands.
      Among the three known pathways that mediate cell migration and are activated by integrins: FAK, PI 3-kinase/AKT, and MAP kinase pathways, we have shown that the FAK (
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ) and the PI 3-kinase/AKT pathways support migration on VN in β3-LNCaP cells and on VN and OPN in PC3 cells. The MAP kinase pathway did not play a role in either β3-LNCaP or PC3 cell migration because PD98059 did not block cell migration (not shown). It should be pointed out that AKT has the ability to support cell migration mediated by vascular endothelial growth factor in endothelial cells (
      • Morales-Ruiz M.
      • Fulton D.
      • Sowa G.
      • Languino L.R.
      • Fujio Y.
      • Walsh K.
      • Sessa W.C.
      ); it is not known, however, whether this mechanism is active in other cells. We show that αvβ3-OPN interaction mediates FAK tyrosine phosphorylation but this signal, although necessary, is not sufficient to mediate cell migration in noninvasive cells. PI 3-kinase, a mediator of integrin and growth factor activities including EGF (
      • Wennstrom S.
      • Downward J.
      ), is known to act as a downstream effector of FAK and to control cell migration activated by cell adhesion to extracellular matrix proteins (
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ,
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ). Integrin-mediated adhesion to the extracellular matrix proteins stimulates the association of the p85 regulatory PI 3-kinase subunit with FAK through FAK Tyr397 (
      • Chen H.-C.
      • Guan J.-L.
      ,
      • Chen H.-C.
      • Guan J.-L.
      ); FAK binding to PI 3-kinase has been demonstrated to activate the latter one (
      • Chen H.-C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.-L.
      ). Because FAK is tyrosine-phosphorylated in response to OPN adhesion mediated by EGF, we conclude that a block at the level of PI 3-kinase/AKT activation downstream of FAK explains the failure of β3-LNCaP cells to migrate, although αvβ3 and the PI 3-kinase/AKT pathway are fully functional in these cells upon αvβ3engagement by VN. The data suggest that the generated β3-LNCaP cells are a model system that allows the study of the αvβ3 effectors that mediate cell migration downstream of FAK. It remains to be analyzed whether FRNK, a negative regulator of FAK that we have shown inhibits VN-mediated migration in β3-LNCaP cells (
      • Zheng D.Q.
      • Woodard A.S.
      • Fornaro M.
      • Tallini G.
      • Languino L.R.
      ), specifically inhibits FAK/PI 3-kinase interaction. It should be stressed that the PI 3-kinase/AKT pathway might also control cell adhesion, as shown by Byzova and Plow since in this study (
      • Byzova T.V.
      • Kim W.
      • Miduar R.J.
      • Plow E.F.
      ) wortmannin did inhibit both cell adhesion and migration after a 30-min preincubation; however, we did not observe wortmannin inhibition of cell adhesion to VN and OPN in our system due to either a cell type-specific effect or to the lack of preincubation with wortmannin in our experimental system. PTEN, a lipid phosphatase that prevents FAK and PI 3-kinase/AKT pathway activation (
      • Tamura M.
      • Gu J.
      • Danen E.H.J.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ,
      • Cairns P.
      • Okami K.
      • Halachmi S.
      • Halachmi N.
      • Esteller M.
      • Herman J.G.
      • Jen J.
      • Isaacs W.B.
      • Bova G.S.
      • Sidransky D.
      ) and down-regulates cell motility and directionality (
      • Gu J.
      • Tamura M.
      • Pankov R.
      • Danen E.H.
      • Takino T.
      • Matsumoto K.
      • Yamada K.M.
      ) is not expected to contribute to the migration of these cells, because LNCaP and PC3 cells have been shown to express a mutated and a deleted PTEN, respectively (
      • Davies M.A.
      • Koul D.
      • Dhesi H.
      • Berman R.
      • McDonnell T.J.
      • McConkey D.
      • Yung W.K.
      • Steck P.A.
      ). In both cell types, PI 3-kinase/AKT pathway activation is controlled by integrins in the absence of an active PTEN, confirming that other molecules such as either Cdc42 (
      • Clark E.A.
      • King W.G.
      • Brugge J.S.
      • Symons M.
      • Hynes R.O.
      ) or ILK (
      • Delcommenne M.
      • Tan C.
      • Gray V.
      • Rue L.
      • Woodgett J.
      • Dedhar S.
      ) could control integrin-mediated cell migration.
      We show here that in β3-LNCaP cells a differential activation of the PI 3-kinase/AKT pathway by αvβ3 occurs: OPN interaction with αvβ3 does not activate the PI 3-kinase/AKT pathway, whereas VN does. It should be noted that PI 3-kinase/AKT pathway is stimulated via OPN engagement of αvβ3 in PC3 cells (Fig. 8 A) and in osteoclasts (
      • Hruska K.A.
      • Rolnick F.
      • Huskey M.
      • Alvarez U.
      • Cheresh D.
      ), thus indicating that the specific failure to activate the PI 3-kinase pathway is cell type-dependent. Because OPN-null mice generate significantly smaller metastases than wild type mice (
      • Crawford H.C.
      • Matrisian L.M.
      • Liaw L.
      ), it is thus conceivable that in the event LNCaP or another noninvasive cell will migrate to a metastatic site where OPN is predominantly expressed, the interaction of αvβ3 with OPN will not provide a migratory or, alternatively, survival signal for these cells. Recently, the role of AKT in promoting cell survival of androgen-sensitive LNCaP cells but not of androgen-insensitive PC3 cells has been shown (
      • Lin J.
      • Adam R.M.
      • Santiestevan E.
      • Freeman M.R.
      ). It is noted that because AKT activation promotes cell survival (
      • Rameh L.E.
      • Cantley L.C.
      ) and LNCaP cells undergo apoptosis in the presence of the PI 3-kinase inhibitor, wortmannin (
      • Lin J.
      • Adam R.M.
      • Santiestevan E.
      • Freeman M.R.
      ), the failure of OPN to stimulate AKT activation might result in apoptosis of these poorly tumorigenic cells. It remains to be determined whether direct αvβ3 integrin engagement in prostate cells prevents apoptosis by activation of the PI 3-kinase/AKT pathway. The synergistic activity of αvβ3, OPN, and the downstream PI 3-kinase/AKT pathway might balance proliferative, apoptotic, and migratory stimuli, thus playing a crucial role in tumor growth and metastatic events in vivo.

      Acknowledgments

      We would like to thank Drs. D. R. Senger, E. A. Wayner, and D. A. Cheresh for generously providing OPN or antibodies. Special thanks to Drs. J. A. Madri and M. Centrella for constructive discussion. We also would like to thank N. Bennett for helping with preparation of the manuscript.

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