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Syk, a Protein-tyrosine Kinase, Suppresses the Cell Motility and Nuclear Factor κB-mediated Secretion of Urokinase Type Plasminogen Activator by Inhibiting the Phosphatidylinositol 3′-Kinase Activity in Breast Cancer Cells*

  • Ganapati H. Mahabeleshwar
    Affiliations
    From the National Centre for Cell Science (NCCS), NCCS Complex, Pune 411 007, India
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  • Gopal C. Kundu
    Correspondence
    To whom correspondence should be addressed. Tel.: 91-20-569-0931 (ext. 203); Fax: 91-20-569-2259;
    Affiliations
    From the National Centre for Cell Science (NCCS), NCCS Complex, Pune 411 007, India
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  • Author Footnotes
    * This work was supported by funds from the Department of Biotechnology (to the National Center for Cell Science) and by an extramural fund from the Department of Biotechnology of the government of India (to G. C. K.).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.
Open AccessPublished:December 10, 2002DOI:https://doi.org/10.1074/jbc.M208905200
      Tumor growth and metastasis are multifaceted processes that mainly involve cell adhesion, proteolytic degradation of the extracellular matrix, and cell migration. Syk is a member of a tyrosine kinase family that is expressed mostly in hematopoietic cells. Syk is expressed in cell lines of epithelial origin, but its function in these cells remains unknown. Here we report that Syk is expressed in MCF-7 cells but not in MDA-MB-231 cells. The overexpression of wild type Syk kinase but not kinase-negative Syk suppressed cell motility and inhibited the activation of phosphatidylinositol (PI) 3′-kinase in MDA-MB-231 cells. In contrast, when Syk-specific antisense S-oligonucleotide but not the sense S-oligonucleotide was transfected to MCF-7 cells the level of PI 3′-kinase activity as well as cell motility were increased. The MDA-MB-231 cells transfected with wild type Syk cDNA followed by treatment with piceatannol, a Syk inhibitor, enhanced cell motility and PI 3′-kinase activity. Pervanadate, a phosphotyrosine phosphatase inhibitor, induced PI 3′-kinase activity and stimulated the interaction between the inhibitor of nuclear factor κBα (IκBα) and the p85α domain of PI 3′-kinase through tyrosine phosphorylation of the IκBα, which ultimately resulted in nuclear factor κB (NFκB) activation. Pervanadate had no effect on the activation of Syk in these cells. However, Syk suppressed the NFκB transcriptional activation and interaction between IκBα and PI 3′-kinase by inhibiting the tyrosine phosphorylation of IκBα. Syk, PI 3′-kinase inhibitors, and NFκB inhibitory peptide inhibited urokinase type plasminogen activator (uPA) secretion and cell motility in these cells. To our knowledge, this is the first report that Syk suppresses the cell motility and inhibits the PI 3′-kinase activity and uPA secretion by blocking NFκB activity through tyrosine phosphorylation of IκBα. These data further demonstrate a functional molecular link between Syk-regulated PI 3′-kinase activity and NFκB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.
      Cell migration and extracellular matrix invasion are two of the major steps in embryonic development (
      • Strickland S.
      • Riech E.
      • Sherman M.I.
      ,
      • Sappiono A.P.
      • Huarte D.
      • Vassali J.D.
      ), wound healing, and cancer cell metastasis (
      • Liotta L.A.
      • Steeg P.S.
      • Stetler-Stevenson W.G.
      ,
      • Testa J.E.
      • Quigley J.P.
      ). However, the exact molecular mechanisms that regulate these processes are not well understood. Syk, a nonreceptor protein-tyrosine kinase, is expressed widely in hematopoietic cells (
      • Kurosaki T.
      ,
      • Turner M.
      • Schweighoffer E.
      • Colucci F.
      • Di Santo J.P.
      • Tybulewicz V.L.
      ). It has tandem amino-terminal SH2
      The abbreviations used are: SH2 domain, Src homology 2 domain; ASSyk, Syk-specific antisense phosphorothioate oligonucleotide(s); FITC, fluorescein isothiocyanate; IκBα, inhibitor of nuclear factor-κB; Luc, luciferase; NFκB, nuclear factor-κB; PI 3′-kinase, phosphatidylinositol 3′-kinase; PIP, phosphatidylinositol phosphate; PMA, phorbol 12-myristate 13-acetate; pV, pervanadate; SSyk, Syk-specific sense phosphorothioate oligonucleotide(s); SykK, kinase-negative Syk(s); TRITC, tetramethylrhodamine isothiocyanate; uPA, urokinase type plasminogen activator
      1The abbreviations used are: SH2 domain, Src homology 2 domain; ASSyk, Syk-specific antisense phosphorothioate oligonucleotide(s); FITC, fluorescein isothiocyanate; IκBα, inhibitor of nuclear factor-κB; Luc, luciferase; NFκB, nuclear factor-κB; PI 3′-kinase, phosphatidylinositol 3′-kinase; PIP, phosphatidylinositol phosphate; PMA, phorbol 12-myristate 13-acetate; pV, pervanadate; SSyk, Syk-specific sense phosphorothioate oligonucleotide(s); SykK, kinase-negative Syk(s); TRITC, tetramethylrhodamine isothiocyanate; uPA, urokinase type plasminogen activator
      domains and a carboxyl-terminal kinase domain (
      • Taniguchi T.
      • Kobayashi T.
      • Kondo J.
      • Takahashi K.
      • Nakamura H.
      • Suzuki J.
      • Nagai K.
      • Yamada T.
      • Nakamura S.
      • Yamamura H.
      ,
      • Yanagi S.
      • Kurosaki T.
      • Yamamura H.
      ). The SH2 domains bind phosphorylated immunoreceptor tyrosine-based activation motifs and play significant roles in signaling through immunoreceptors (
      • Cheng A.M.
      • Negishi I.
      • Anderson S.J.
      • Chan A.C.
      • Bolen J.
      • Loh D.Y.
      • Pawson T.
      ). ZAP-70 is a cytoplasmic tyrosine kinase, and both Syk and ZAP-70 share the same tandem SH2 domains at the amino terminus and play important roles in coupling antigen and Fc receptors to downstream signaling events that mediate diverse cellular responses including proliferation, differentiation, and phagocytosis (
      • Cheng A.M.
      • Negishi I.
      • Anderson S.J.
      • Chan A.C.
      • Bolen J.
      • Loh D.Y.
      • Pawson T.
      ,
      • Indik Z.K.
      • Park J.G.
      • Pan X.Q.
      • Schreiber A.D.
      ). Fc receptors are members of the family of membrane proteins, called immunoreceptors, and they are expressed in all cells of the immune system. Both Syk and ZAP-70 are regulated by β3 integrin-dependent cell adhesion via phosphorylation-independent interaction with the cytoplasmic domain of β3 integrin (
      • Woodside D.G.
      • Obergfell A.
      • Talapatra A.
      • Calderwood D.A.
      • Shattil S.J.
      • Ginsberg M.H.
      ). The expression of Syk has also been reported in cell lines of epithelial origin (
      • Fluck M.
      • Zurcher G.
      • Andres A.C.
      • Ziemiecki A.
      ), but its function in these cells is not well understood. Recently it has been documented that Syk is commonly expressed in normal human breast tissue, benign breast lesions, and low tumorigenic breast cancer cell lines (
      • Coopmen P.J.P.
      • Do M.T.H.
      • Barth M.
      • Bowden E.T.
      • Hayes A.J.
      • Basyuk E.
      • Blancato J.K.
      • Vezza P.R.
      • McLeskey S.W.
      • Mangeat P.H.
      • Mueller S.C.
      ).
      Several cytokines, growth factors, and other agents control the regulation of cell motility. Phosphatidylinositol 3′-kinase (PI 3′-kinase) also plays significant role in regulation of cell motility (
      • Derman M.P.
      • Toker A.
      • Hartwig J.H.
      • Spokes K.
      • Falck J.R.
      • Chen C.-S.
      • Cantley L.C.
      • Cantley L.G.
      ). Two subunits are present among all of the classes of PI 3′-kinases. One is catalytic subunit p110 (α, β, δ) or p110γ, and the other is regulatory subunit p85 (α, β, p55γ, and p101) (
      • Domin J.
      • Waterfield M.D.
      ). The regulatory subunit of PI 3′-kinase is responsible for B cell development and proliferation (
      • Suzuki H.
      • Terauchi Y.
      • Fujiwara M.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      • Koyasu S.
      ), and the catalytic subunits are critical for chemotactic activity (
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Waterfield M.D.
      • Ridley A.J.
      ).
      The activation of NFκB is regulated by a number of proinflammatory stimuli (
      • Bauerle P.A.
      • Baltimore D.
      ,
      • Baldwin A.S.
      ). The NFκB family consists of several members including p65, p50, RelB, and c-Rel molecules (
      • Bauerle P.A.
      • Baltimore D.
      ). The activity of NFκB is also tightly controlled by its inhibitor, IκB family of proteins (
      • Karin M.
      ). NFκB forms a complex with IκBα, and the complex can be removed from the nucleus by exportin-mediated transport to the cytoplasm. Recent report indicated that constitutively active PI 3′-kinase controls the activation of NFκB by the association of tyrosine-phosphorylated IκBα with the regulatory subunit of PI 3′-kinase, p85α (
      • Beraud C.M.
      • Henzel W.J.
      • Baeuerle P.A.
      ). The recent data also demonstrated that interleukin-1 stimulates PI 3′-kinase dependent phosphorylation and transactivation of NFκB without nuclear translocation of NFκB, indicating an alternative pathway other than IκBα-mediated pathway (
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ). But the molecular mechanism by which Syk, a tyrosine kinase, regulates the PI 3′-kinase dependent activation of NFκB in breast cancer cells is not well defined.
      Urokinase-type plasminogen activator (uPA) is a member of the serine protease family which induces the conversion of plasminogen to plasmin (
      • Besser D.
      • Verde P.
      • Nagamine Y.
      • Blasi F.
      ). Plasmin regulates cell invasion by degrading matrix proteins such as fibronectin, type IV collagen, and laminin or indirectly by activating matrix metalloproteinases and uPA (
      • Sheela S.
      • Barrett J.C.
      ,
      • Dano K.
      • Andreasen P.A.
      • Grondahl-Hansen J.
      • Kristensen P.
      • Nielsen L.S.
      • Skriver L.
      ,
      • Murphy G.
      • Willenbrock F.
      • Crabbe T.
      • O'Shea M.
      • Ward R.
      • Atkinson S.
      • O'Connell J.
      • Docherty A.
      ). Previous reports shown that uPA plays a significant role in tumor growth and metastasis. The signaling pathway by which Syk controls uPA secretion through PI 3′-kinase-dependent activation of NFκB is not clearly understood.
      In this study, we demonstrate that overexpression of wild type Syk in MDA-MB-231 cells suppressed cell motility and reduced PI 3′-kinase activity. Syk-specific antisense phosphorothioate oligonucleotide (ASSyk), when transfected to MCF-7 cells, increased cell motility and up-regulated PI 3′-kinase activity. Pervanadate (pV) stimulated PI 3′-kinase activity and induced transactivation of NFκB through tyrosine phosphorylation of IκBα, whereas Syk down-regulated the NFκB activity by inhibiting tyrosine phosphorylation of IκBα in these cells. Syk, PI 3′-kinase inhibitor, and NFκB inhibitors inhibited cell motility and uPA secretion in these cells. Taken together, Syk suppressed cell motility, uPA secretion, and PI 3′-kinase-mediated NFκB activation by inhibiting the interaction between the p85α subunits of PI 3′-kinase and tyrosine-phosphorylated IκBα.

      DISCUSSION

      In this study, we have demonstrated that Syk, a nonreceptor protein-tyrosine kinase, inhibited PI 3′-kinase activity and subsequently suppressed cell motility in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. Syk is expressed in MCF-7 cells, but its expression is not detectable in highly invasive MDA-MB-231 cells. The wild type Syk cDNA was transfected in MDA-MB-231 cells, and its expression and autophosphorylation activity in these cells were comparable with the endogenous Syk activity in MCF-7 cells (data not shown). Furthermore, we have demonstrated that Syk suppresses the pV-induced interaction of p85 subunit of PI 3′-kinase and tyrosine-phosphorylated IκBα. Syk reduced the NFκB luciferase activity and piceatannol, a Syk inhibitor, enhanced the NFκB activity in both MCF-7 and MDA-MB-231 cells. The inhibition of PI 3′-kinase activity down-regulates the NFκB transactivation as well as cell motility in these cells. Moreover, inhibition of PI 3′-kinase and NFκB activities by their specific inhibitors reduced uPA secretion and cell motility in these cells. Syk also inhibits the uPA secretion in both MCF-7 and MDA-MB-231 cells. These data demonstrated that Syk suppresses cell motility and down-regulates NFκB activity by inhibiting the PI 3′-kinase activity and uPA secretion in both MCF-7 and MDA-MB-231 cells.
      ZAP-70, a nonreceptor protein-tyrosine kinase that shares the same tandem SH2 domains with Syk at the amino terminus, was not detected in the breast cancer cells (data not shown). Previous reports have indicated that pV, a phosphotyrosine phosphatase inhibitor, induces autophosphorylation of Syk in MCF-7 cells (
      • Coopmen P.J.P.
      • Do M.T.H.
      • Barth M.
      • Bowden E.T.
      • Hayes A.J.
      • Basyuk E.
      • Blancato J.K.
      • Vezza P.R.
      • McLeskey S.W.
      • Mangeat P.H.
      • Mueller S.C.
      ). However, our data revealed that pV had no effect on activation of Syk in MCF-7 cells. Earlier reports have shown that PI 3′-kinase signaling is required for depolarization and cell migration of MCF-7 cells by insulin-like growth factor I (
      • Guvakova M.A.
      • Surmacz E.
      ). The data also suggested that increased PI 3′-kinase activity is correlated with the migratory potential and metastatic activity of highly invasive breast cancer (MDA-MB-231) cells (
      • Sliva D.
      • Rizzo M.T.
      • English D.
      ). Using genetic (wild type Syk and SykK) and pharmacological (piceatannol) inhibitors of Syk, we have demonstrated that Syk is involved in the suppression of cell motility, PI 3′-kinase activity, and NFκB-mediated uPA secretion in both MCF-7 and MDA-MB-231 cells. The p110 isoforms of PI 3′-kinase played significant roles in cell migration, and differential activation of specific p110 isoforms is responsible for particular signaling events in different cell types (
      • Sasaki T.
      • Irie-Sasaki J.
      • Jones R.G.
      • Oliviera-dos-Santos A.J.
      • Stanford W.L.
      • Bolon B.
      • Wakeham A.
      • Itie A.
      • Bouchard D.
      • Kozieradzki I.
      • Joza N.
      • Mak T.W.
      • Ohashi P.S.
      • Suzuki A.
      • Penninger J.M.
      ,
      • Hill K.
      • Welti S.
      • Yu J.
      • Murray J.T.
      • Yip S.-C.
      • Condeelis J.S.
      • Segall J.E.
      • Backer J.M.
      ). Recently, Sliva et al. (
      • Sliva D.
      • Rizzo M.T.
      • English D.
      ) reported that the regulatory p85α subunit of PI 3′-kinase is essential for enhanced migration of metastatic tumor cells because overexpression of a dominant negative regulatory subunit (p85DN) drastically reduced the cell migration.
      It has been documented recently that PI 3′-kinase plays a significant role in NFκB activation in different cell types (
      • Reddy S.A.G.
      • Huang J.H.
      • Liao W.S.-L.
      ,
      • Pan Z.K.
      • Christiansen S.C.
      • Ptasznik A.
      • Zuraw B.L.
      ). Tumor necrosis factor α-induced NFκB activation is not affected by PI 3′-kinase inhibitors (wortmannin and LY294002). Similarly, a PI 3′-kinase inhibitor has no effect on interleukin-1-dependent IκBα degradation, nuclear translocation of NFκB, and NFκB-DNA binding (
      • Sizemore N.
      • Leung S.
      • Stark G.R.
      ). Our results demonstrated that Syk down-regulates NFκB transactivation by inhibiting the interaction between the tyrosine-phosphorylated IκBα and the p85 subunit of PI 3′-kinase. Earlier reports have shown that pV and tumor necrosis factor α induced NFκB activation in Jurkat cells, and only pV-induced activation of NFκB is inhibited by wortmannin (
      • Beraud C.M.
      • Henzel W.J.
      • Baeuerle P.A.
      ). Both wortmannin and LY294002 abrogated the transactivation of NFκB but had no effect on NFκB-DNA binding in MDA-MB-231 cells (
      • Sliva D.
      • Rizzo M.T.
      • English D.
      ). Our data also revealed that Syk and PI 3′-kinase inhibitors down-regulate the transactivation of NFκB but not NFκB-DNA binding (data not shown) in both MCF-7 and MDA-MB-231 cells. Thus we conclude that Syk-regulated transactivation of NFκB is independent of NFκB-DNA binding in breast cancer cells.
      We have shown that MDA-MB-231 cells transfected with wild type Syk but not with SykK suppressed the cellular migration and treatment of wild type Syk-transfected cells with increasing concentrations of piceatannol enhanced the cell migration in these cells. Both wortmannin and LY294002 inhibited, but pV induced the migration of MCF-7 and MDA-MB-231 cells. The pV-induced migration is blocked by wortmannin or LY294002 in these cells. Similarly, the NFκB inhibitory peptide SN-50, curcumin, and protein synthesis inhibitor (actinomycin-D) reduced cell migration. Pretreatment of nontransfected or Syk-transfected cells with anti-uPA antibody reduced the migration of these cells. These data suggested that Syk suppressed the cell migration by down-regulating the constitutively active NFκB activation and uPA secretion by inhibiting the PI 3′-kinase activity in breast cancer cells.
      Our data also revealed that pV induces tyrosine phosphorylation of IκBα and subsequently enhances the interaction between tyrosine-phosphorylated IκBα with the p85α domain of PI 3′-kinase in a time-dependent manner in both cell lines. ST 638, a tyrosine kinase inhibitor, blocked the pV-induced tyrosine phosphorylation of IκBα. However, pV had no effect on serine phosphorylation of IκBα in these cells, suggesting that pV-induced transactivation of NFκB occurs through tyrosine phosphorylation of IκBα. Syk suppressed the tyrosine phosphorylation of IκBα and reduced the interaction between tyrosine-phosphorylated IκBα and p85 subunit of PI 3′-kinase. These data demonstrated that Syk regulates the transactivation of NFκB by inhibiting the direct interaction of tyrosine-phosphorylated IκBα and the p85 domain of PI 3′-kinase and suggested an alternative pathway not involving the phosphorylation and degradation of IκBα pathways.
      Matrix metalloproteinases play a major role in the regulation of cancer cell migration, extracellular matrix invasion, and metastasis by degrading the extracellular matrix proteins (
      • Liotta L.A.
      • Steeg P.S.
      • Stetler-Stevenson W.G.
      ,
      • Testa J.E.
      • Quigley J.P.
      ,
      • Murphy G.
      • Gavrilovic J.
      ). We and others have shown recently that NFκB plays significant roles in the activation of matrix metalloproteinases-1, -2, -3, and -9 (
      • Philip S.
      • Bulbule A.
      • Kundu G.C.
      ,
      • Hansen S.K.
      • Nerlov C.
      • Zabel U.
      • Verde P.
      • Johnsen M.
      • Baeuerle P.A.
      • Blasi F.
      ,
      • Bond M.
      • Baker A.H.
      • Newby A.C.
      ). uPA is also responsible for the migration and regulation of matrix metalloproteinases activation through NFκB-mediated pathways (
      • Bond M.
      • Fabunmi R.P.
      • Baker A.H.
      • Newby A.C.
      ). In this study, we have detected the level of uPA in both highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The constitutive secretion of uPA is significantly higher in MDA-MB-231 cells; however, a low level of uPA expression is observed in MCF-7 cells. The data also indicate that Syk down-regulates whereas pV up-regulates the uPA production in these cells. The PI 3′-kinase inhibitors wortmannin and LY294002 or the NFκB inhibitor SN-50 reduced uPA secretion, especially in MDA-MB-231 cells.
      In summary, we have demonstrated for the first time that overexpression of wild type Syk kinase but not the SykK suppresses cell motility and reduces the activation of PI 3′-kinase in MDA-MB-231 cells. In contrast, in ASSyk but not SSyk, when transfected to the MCF-7 cells, the level of PI 3′-kinase activity as well as cell motility were increased. In the wild type Syk cDNA-transfected MDA-MB-231 cells, when treated with piceatannol, a Syk inhibitor, PI 3′-kinase activity increased. pV, a tyrosine phosphatase inhibitor, enhances the activity of PI 3′-kinase and induces the interaction between p85α, the regulatory subunit of PI 3′-kinase, and IκBα through tyrosine phosphorylation of IκBα. Syk suppresses the transactivation of NFκB by inhibiting the tyrosine phosphorylation of IκBα. PI 3′-kinase inhibitor reduces the pV-induced transactivation of NFκB. Both PI 3′-kinase inhibitors wortmannin and LY294002 and NFκB inhibitory peptide SN-50 suppress cell motility, indicating that PI 3′-kinase and NFκB play significant roles in this process. ASSyk-transfected MCF-7 cells enhanced uPA secretion, whereas wild type Syk cDNA-transfected MDA-MB-231 cells reduced uPA production, indicating that Syk down-regulates uPA secretion. These data suggested that Syk suppresses the NFκB transactivation by inhibiting the direct interaction of tyrosine-phosphorylated IκBα with the p85α domain of PI 3′-kinase in breast cancer cells. Finally, these data demonstrated that Syk down-regulates PI 3′-kinase activity and suppresses the constitutive NFκB activity and uPA secretion that ultimately lead to the suppression of cell motility of breast cancer cells (Fig. 9). These findings may be useful in designing novel therapeutic interventions using Syk as a target molecule that will disrupt the PI 3′-kinase and NFκB signaling pathways, resulting in reduction of uPA secretion and consequent blocking of invasiveness, migration, and metastatic spread of breast cancer.
      Figure thumbnail gr9
      Figure 9Molecular mechanism of Syk-regulated NFκB activation and uPA secretion through activation of PI 3′-kinase in breast cancer cells. Syk suppresses cell motility and PI 3′-kinase activity in breast cancer cells. Syk also inhibits NFκB activation by blocking the interaction between p85α subunits of PI 3′-kinase and tyrosine-phosphorylated IκBα and subsequently reduces the uPA secretion in these cells. Wortmannin, LY294002, SN-50, and curcumin specifically disrupt these signaling pathways.

      Acknowledgments

      We thank Dr. Susette C. Mueller for providing wild type and SykK cDNAs and Dr. Rainer de Martin for providing the pNFκB-Luc containing five tandem repeats of the NFκB binding site. We also thank Riku Das and Hema Rangaswami for critically reading this manuscript.

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