Vascular Endothelial Growth Factor-induced Migration of Multiple Myeloma Cells Is Associated with β1 Integrin- and Phosphatidylinositol 3-Kinase-dependent PKCα Activation

In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and β1integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) α is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify β1 integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and β1 integrin. Moreover, we demonstrate that PKCα is constitutively associated with β1 integrin. These data are consistent with PKCα-dependent exocytosis of activated β1 integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKCα-driven internalization of β1 integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and β1 integrin-mediated binding to fibronectin are required for the recruitment and activation of PKCα. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin. In multiple myeloma (MM), migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion within the BM microenvironment, and for egress into the peripheral blood. In the present study we characterize the role of vascular endothelial growth factor (VEGF) and β1integrin (CD29) in MM cell migration. We show that protein kinase C (PKC) α is translocated to the plasma membrane and activated by adhesion of MM cells to fibronectin and VEGF. We identify β1 integrin modulating VEGF-triggered MM cell migration on fibronectin. We show that transient enhancement of MM cell adhesion to fibronectin triggered by VEGF is dependent on the activity of both PKC and β1 integrin. Moreover, we demonstrate that PKCα is constitutively associated with β1 integrin. These data are consistent with PKCα-dependent exocytosis of activated β1 integrin to the plasma membrane, where its increased surface expression mediates binding to fibronectin; conversely, catalytically active PKCα-driven internalization of β1 integrin results in MM cell de-adhesion. We show that the regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85) is constitutively associated with FMS-like tyrosine kinase-1 (Flt-1). VEGF stimulates activation of PI 3-kinase, and both MM cell adhesion and migration are PI 3-kinase-dependent. Moreover, both VEGF-induced PI 3-kinase activation and β1 integrin-mediated binding to fibronectin are required for the recruitment and activation of PKCα. Time-lapse phase contrast video microscopy (TLVM) studies confirm the importance of these signaling components in VEGF-triggered MM cell migration on fibronectin. multiple myeloma bone marrow vascular endothelial growth factor phosphatidylinositol VEGF receptor protein kinase C FMS-like tyrosine kinase very late antigens extracellular matrix antibody monoclonal Ab fetal bovine serum bisindolylmaleimide I Ca-dependent tyrosine kinase phorbol-12-myristate-13-acetate phospholipase C γ immunoprecipitation In multiple myeloma (MM)1 migration is necessary for the homing of tumor cells to bone marrow (BM), for expansion of malignant plasma cells within the BM microenvironment, and for the egress into peripheral blood. It has been reported that the extracellular matrix (ECM) proteins laminin, microfibrillar collagen type VI, and fibronectin are strong adhesive components for MM cells and that adhesion to laminin and fibronectin is β1integrin (CD29)-mediated (1Kibler C. Schermutzki F. Waller H.D. Timpl R. Muller C.A. Klein G. Cell Adhes. Commun. 1998; 5: 307-323Crossref PubMed Google Scholar). β1 integrins are typically expressed on MM cells, specifically α integrins VLA-4 (α4β1) and VLA-5 (α5β1) (2Damiano J.S. Cress A.E. Hazlehurst L.A. Shtil A.A. Dalton W.S. Blood. 1999; 93: 1658-1667Crossref PubMed Google Scholar, 3Jensen G.S. Belch A.R. Mant M.J. Ruether B.A. Yacyshyn B.R. Pilarski L.M. Am. J. Hematol. 1993; 43: 29-36Crossref PubMed Scopus (57) Google Scholar). β1integrin-mediated adhesion of MM cells to fibronectin confers protection against drug-induced apoptosis and triggers NFκB-dependent transcription and secretion of interleukin-6, the major MM growth and survival factor (4Uchiyama H. Barut B.A. Chauhan D. Cannistra S.A. Anderson K.C. Blood. 1992; 80: 2306-2314Crossref PubMed Google Scholar, 5Uchiyama H. Barut B.A. Mohrbacher A.F. Chauhan D. Anderson K.C. Blood. 1993; 82: 3712-3720Crossref PubMed Google Scholar). Interestingly, chimeric mice (β1−/− → wild-type chimeras) lack β1-null cells in blood and in hematopoietic organs such as spleen, thymus, and BM as a consequence of the inability of β1-null cells to invade the fetal liver (6Fassler R. Meyer M. Genes Dev. 1995; 9: 1896-1908Crossref PubMed Scopus (612) Google Scholar). In addition to up-regulation of cell surface expression and induction of surface-clustering, integrin activity can be triggered by multiple agonists through “inside-out” signaling independent of changes in integrin expression levels (e.g. ligand binding to growth factor receptors is associated with changes in the way in which adhesion receptors on the cell surface engage the ECM). This concept is illustrated in human umbilical vein endothelial cells in which VEGF stimulates β1 integrins and leads to markedly enhanced movement (7Byzova T.V. Goldman C.K. Pampori N. Thomas K.A. Bett A. Shattil S.J. Plow E.F. Mol. Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar). Although VEGF induces migration as a key step in angiogenesis, the interplay between VEGF and integrins is not restricted to angiogenesis. VEGF and VEGFR are expressed by many tumor cell lines; moreover, elevated levels of VEGF are found in cancer patients, and inhibition of VEGF can suppress tumor growth (8Ferrara N. J. Mol. Med. 1999; 77: 527-543Crossref PubMed Scopus (1077) Google Scholar). Indeed, clinical studies are underway investigating VEGF as a novel therapeutic target (9Ferrara N. Alitalo K. Nat. Med. 1999; 5: 1359-1364Crossref PubMed Scopus (915) Google Scholar). In MM VEGF is expressed and secreted by tumor cells as well as BM stromal cells (10Bellamy W.T. Richter L. Frutiger Y. Grogan T.M. Cancer Res. 1999; 59: 728-733PubMed Google Scholar, 11Dankbar B. Padro T. Leo R. Feldmann B. Kropff M. Mesters R.M. Serve H. Berdel W.E. Kienast J. Blood. 2000; 95: 2630-2636Crossref PubMed Google Scholar); moreover, binding of MM cells to BM stromal cells enhances both interleukin-6 and VEGF secretion (11Dankbar B. Padro T. Leo R. Feldmann B. Kropff M. Mesters R.M. Serve H. Berdel W.E. Kienast J. Blood. 2000; 95: 2630-2636Crossref PubMed Google Scholar). We recently showed that in addition to stimulating angiogenesis, VEGF directly induces MM cell proliferation via a protein kinase C (PKC)-independent mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway and triggers MM cell migration on fibronectin via a PKC-dependent pathway (12Podar K. Tai Y.T. Davies F.E. Lentzsch S. Sattler M. Hideshima T. Lin B.K. Gupta D. Shima Y. Chauhan D. Mitsiades C. Raje N. Richardson P. Anderson K.C. Blood. 2001; 98: 428-435Crossref PubMed Scopus (381) Google Scholar). Members of the PKC family mediate multiple physiological functions (13Asaoka Y. Nakamura S. Yoshida K. Nishizuka Y. Trends Biochem. 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By showing that VEGF-mediated MM cell migration is associated with β1 integrin- and PI 3-kinase-dependent PKCα activation, we further confirm the importance of tumor cell-BM microenvironment interaction as a pivotal process in the pathogenesis of MM. Moreover, our studies identify several potential targets for novel therapies to improve outcome in MM. Recombinant human VEGF165 was purchased from R 79: 213-222Crossref PubMed Google Scholar) was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 10 μg/ml streptomycin, and 2 mml-glutamine. Cells were starved for 15–18 h in medium with 3% and for h to with VEGF human R Full Text PDF PubMed Scopus Google Scholar) in a of The was and on a These were the cell adhesion to the in MM.1S cells were and with cell for with RPMI 1640 the cells were with β1 integrin bisindolylmaleimide I and with VEGF. The cell were to cells were by with RPMI 1640 by of the cells were in a and were in Cell migration was as (12Podar K. Tai Y.T. Davies F.E. Lentzsch S. Sattler M. Hideshima T. Lin B.K. Gupta D. Shima Y. Chauhan D. Mitsiades C. Raje N. Richardson P. Anderson K.C. Blood. 2001; 98: 428-435Crossref PubMed Scopus (381) Google Scholar, B. L.A. B.R. J. Cell Biol. 1995; PubMed Scopus Google Scholar, M. Cell Res. 1994; PubMed Scopus Google Scholar). cells that into the of a were a MM.1S cells were starved in RPMI medium for h and to in the of VEGF with with a and was to video CA). and to a were the were with the To migration the of the of cells on were The was based on the of by the of of cells was for of in was The of was we showed that VEGF-triggered MM cell migration on fibronectin is mediated via a PKC-dependent signaling pathway (12Podar K. Tai Y.T. Davies F.E. Lentzsch S. Sattler M. Hideshima T. Lin B.K. Gupta D. Shima Y. Chauhan D. Mitsiades C. Raje N. Richardson P. Anderson K.C. Blood. 2001; 98: 428-435Crossref PubMed Scopus (381) Google Scholar). In the present study we and characterize the interrelationship of and in PKC that leads to MM cell migration. PKC including PKCα N. C. D. D. H. M. J. 1998; PubMed Scopus Google K.J. M. S. Cancer Res. 1999; 59: Google and S. Parker C. J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have been in a cell of PKCα was to cell of cancer cells (26Ng T. Shima D. Squire A. Bastiaens P.I. Gschmeissner S. Humphries M.J. Parker P.J. EMBO J. 1999; 18: 3909-3923Crossref PubMed Scopus (281) Google Scholar). We have that MM cell migration is PKC-dependent can be by the PKC I (12Podar K. Tai Y.T. Davies F.E. Lentzsch S. Sattler M. Hideshima T. Lin B.K. Gupta D. Shima Y. Chauhan D. Mitsiades C. Raje N. Richardson P. Anderson K.C. Blood. 2001; 98: 428-435Crossref PubMed Scopus (381) Google Scholar). a step to identify the of PKC VEGF-induced migration in MM cells, we the expression of the PKC isoforms in MM cell and cells that and are expressed in human MM cell and MM cells in contrast to and data not in our studies we the MM.1S human MM cell line (28Moalli P.A. Pillay S. Weiner D. Leikin R. Rosen S.T. Blood. 1992; 79: 213-222Crossref PubMed Google Scholar) as a for the activation of PKC with recruitment to the plasma membrane, we of cell with against and to the VEGF In MM cells PKC isoforms were in the VEGF of cells on PKCα translocated into the membrane 2 changes in of PKC isoforms as and were The activation of PKCα VEGF of cells to fibronectin was further by a in kinase kinase activation by as a 2 Although a protein kinase was not the of a by of cells on fibronectin of cells with VEGF the of PKCα In with a major PKC of PKC and into the membrane as Anderson Nature. PubMed Scopus Google Scholar, Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar) the microfibrillar collagen type VI, and fibronectin MM cells, and adhesion to laminin and fibronectin is β1 integrin (CD29)-mediated (1Kibler C. Schermutzki F. Waller H.D. Timpl R. Muller C.A. Klein G. Cell Adhes. Commun. 1998; 5: 307-323Crossref PubMed Google Scholar). β1 integrins expressed on MM cells VLA-4 (α4β1) and VLA-5 (α5β1) (2Damiano J.S. Cress A.E. Hazlehurst L.A. Shtil A.A. Dalton W.S. Blood. 1999; 93: 1658-1667Crossref PubMed Google Scholar, 3Jensen G.S. Belch A.R. Mant M.J. Ruether B.A. Yacyshyn B.R. Pilarski L.M. Am. J. Hematol. 1993; 43: 29-36Crossref PubMed Scopus (57) Google Scholar, H. Barut B.A. Chauhan D. Cannistra S.A. Anderson K.C. Blood. 1992; 80: 2306-2314Crossref PubMed Google Scholar, 5Uchiyama H. Barut B.A. Mohrbacher A.F. Chauhan D. Anderson K.C. Blood. 1993; 82: 3712-3720Crossref PubMed Google which mediate to both the and BM stromal In human umbilical vein endothelial cells, VEGF stimulates β1 integrins via inside-out to increased (7Byzova T.V. Goldman C.K. Pampori N. Thomas K.A. Bett A. Shattil S.J. Plow E.F. Mol. Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar). migration is a process of cell adhesion and we VEGF can β1integrin-mediated MM cell MM cells to and adhesion was increased with VEGF of VEGF-mediated cell adhesion were fibronectin of adhesion to levels fibronectin VEGF-mediated in adhesion were with binding of VEGF and We the of adhesion of VEGF was and in with the PKC I well as with the VEGF-induced cell adhesion to fibronectin. This of PKC in MM cell adhesion to fibronectin was further by a of adhesion triggered by to that by VEGF. our results show that VEGF enhances MM cell adhesion to dependent on both PKC and β1 integrin We to β1integrin VEGF-mediated MM cell migration on fibronectin. in β1 integrin mAb not mediated inhibition of VEGF-triggered MM cell migration in a These data confirm that β1integrin (CD29) is the integrin associated with VEGF-triggered MM cell migration on fibronectin. The to the of the integrin receptor are of cell movement is to be by the and of integrins through surface the and from the cell D.A. A.F. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In cells (26Ng T. Shima D. Squire A. Bastiaens P.I. Gschmeissner S. Humphries M.J. Parker P.J. EMBO J. 1999; 18: 3909-3923Crossref PubMed Scopus (281) Google Scholar) recently have found that PKCα with activated β1 which its exocytosis to the plasma moreover, catalytically active PKCα is for β1 integrin internalization through a and PI PKCα up-regulation of the cell migration of these cells, which was that the internalization of the receptor We PKCα and β1 integrin are associated in MM between these proteins was by activation and conventional with proteins of the PKC to several of integrins Cell Biol. 1998; PubMed Scopus Google Scholar) including B.A. F. J. 1996; Google Scholar) and E. F. M. M. C. J. 1994; PubMed Scopus Google Scholar). of binding to is dependent on the extracellular domain of the integrin α and the results in of the integrin α G. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). In we are investigating the regulatory role of these proteins on VEGF-induced MM cell migration. In addition to cell via β1 integrin catalytically active PKC also components of the including the tyrosine kinase also as tyrosine kinase 2 and adhesion tyrosine kinase is a tyrosine kinase to adhesion kinase is a kinase for the of signaling and thereby the of surface to the and signaling pathways associated with cell and migration. of studies have tyrosine of in cells of hematopoietic (e.g. and cells, cells, bone marrow cells, and that in these cells are associated with cell (e.g. activation is required for and J.S. D. J.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In MM.1S cells, we have that is activated its role in apoptosis D. Hideshima T. P. S. G. Raje N. Rosen S. N. H. S. S. Anderson K.C. 1999; 18: PubMed Scopus Google Scholar). has been reported to and integrin-mediated signaling to the in and hematopoietic cells S. R. Y. S. D. J. S. L.M. H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. R. P. E. B. J. Nature. 1995; PubMed Scopus Google Scholar, H. K. M. H. K. T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (365) Google Scholar, J. H. S. S. Blood. 1996; PubMed Google Scholar). results show that VEGF induces the of with the β1 protein VEGFR immunoprecipitation and studies and showed We the VEGF-induced of a membrane β1 and Flt-1 changes the activity of the associated in MM in VEGF increased tyrosine in MM.1S of proteins was by and the membrane with The PKC enhanced of VEGF-triggered activation was by both the PKC I and a β1integrin mAb These results that the activation of in MM cells is by both and β1 integrin-mediated signaling Moreover, a that in hematopoietic cells is also associated with the protein R. S. E. S. Sattler M. H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We that VEGF to the in MM cells and MM cell migration adhesion on fibronectin via studies are directed role of in MM cells in We to identify the inside-out signaling components of β1 integrin by VEGF in MM PI and to and thereby and PKC activation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activity is enhanced by both via tyrosine and directly through the binding of to the domain and the 2 of J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. G. J. EMBO J. 1998; 17: PubMed Scopus Google Scholar, Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We have recently that Flt-1 VEGFR is expressed in the human MM cell line and MM cells and is VEGF (12Podar K. Tai Y.T. Davies F.E. Lentzsch S. Sattler M. Hideshima T. Lin B.K. Gupta D. Shima Y. Chauhan D. Mitsiades C. Raje N. Richardson P. Anderson K.C. Blood. 2001; 98: 428-435Crossref PubMed Scopus (381) Google Scholar). Flt-1 and the subunit of PI 3-kinase were associated in cells S.A. T.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google we interaction is in MM Flt-1 was from VEGF and MM.1S cells by with illustrated in is constitutively associated with that PI 3-kinase in VEGF To confirm we PI 3-kinase activation is in VEGF-induced signaling in MM VEGF tyrosine of the regulatory subunit of PI 3-kinase, by of with mAb Moreover, of with mAb showed increased of with which was of VEGF To VEGF-induced the of PI 3-kinase, we in PI as a in VEGF activation of PI 3-kinase activity these data show that activation of PI 3-kinase is in the VEGF-triggered Flt-1 signaling We activation of PI 3-kinase is required for activation of PKCα. PI 3-kinase activation in MM cells on fibronectin was by PKCα recruitment to the plasma membrane was also These results that VEGF-induced PI 3-kinase activation with β1 integrin binding to fibronectin PKCα. To activation of PI 3-kinase is necessary for MM cell we a to the of VEGF on the migration activity of MM.1S cells on with fibronectin as a for in VEGF to the medium in the a migration of growth MM.1S cells on fibronectin in the which was by the PKC with the PI 3-kinase for to VEGF MM cell migration. In VEGF not migration of MM.1S cells on in the We VEGF-induced in adhesion were also PI 3-kinase-dependent. in with the PI 3-kinase VEGF-induced MM cell adhesion to fibronectin. In VEGF not MM cell adhesion these results demonstrate that PI 3-kinase activation MM cell migration on fibronectin These are to the and PI 3-kinase-dependent reported in cancer cells (26Ng T. Shima D. Squire A. Bastiaens P.I. Gschmeissner S. Humphries M.J. Parker P.J. EMBO J. 1999; 18: 3909-3923Crossref PubMed Scopus (281) Google Scholar). is a process of cell adhesion and and both in and To our of the results and to with the changes in MM cell that mediate cell we phase contrast video microscopy (TLVM) and migration and with are the of are the cell In MM.1S cells were on fibronectin in the of VEGF. cells were for h and phase contrast were with the MM cells to to and the movement was increased by VEGF and In MM cells to fibronectin and increased membrane Although with VEGF not membrane the migration was markedly increased in the of the PKC migration was to levels obtained in MM cells to fibronectin of MM cell of MM cells was as and In MM.1S cells were on fibronectin not in the of The of MM cell migration was as and The migration of MM cells on with to the of The for the of and results were cells in the of fibronectin. binding of MM cells to fibronectin a of cell h and with the VEGF in h 100 and with cells to fibronectin. This was to migration in the of 2 This study that VEGF-mediated MM cell migration is associated with β1 integrin- and PI 3-kinase-dependent PKCα To further the role of PKCα as a potential therapeutic target in studies are investigating the of both in and in MM We Dr. (Dana-Farber Cancer Dr. and Dr. for