Abstract Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination. Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells. Tumor cells (CD38+CD45RA−) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v). Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus. EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity. Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity. Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86. Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents. Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.
<p>Supplementary Figure 4. LY2495655 attenuates loss of muscle weight normalized to brain weight but has no effect on brain or heart weight in C26 tumor bearing mice in the presence of gemcitabine.</p>
To compare disease-free survival (DFS) after maintenance therapy with the selective protein kinase C β (PKCβ) inhibitor, enzastaurin, versus placebo in patients with diffuse large B-cell lymphoma (DLBCL) in complete remission and with a high risk of relapse after first-line therapy.This multicenter, phase III, randomized, double-blind, placebo-controlled trial enrolled patients who were at high risk of recurrence after rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients (N = 758) with stage II bulky or stage III to IV DLBCL, three or more International Prognostic Index risk factors at diagnosis, and a complete response or unconfirmed complete response after 6 to 8 cycles of R-CHOP were assigned 2:1 to receive oral enzastaurin 500 mg daily or placebo for 3 years or until disease progression or unacceptable toxicity. Primary end point was DFS 3 years after the last patient entered treatment. Correlative analyses of biomarkers, including cell of origin by immunohistochemistry and PKCβ expression, with efficacy outcomes were exploratory objectives.After a median follow-up of 48 months, DFS hazard ratio for enzastaurin versus placebo was 0.92 (95% CI, 0.689 to 1.216; two-sided log-rank P = .541; 4-year DFS, 70% v 71%, respectively). Independent of treatment, no significant associations were observed between PKCβ protein expression or cell of origin and DFS or overall survival.Enzastaurin did not significantly improve DFS in patients with high-risk DLBCL after achieving complete response to R-CHOP. Achievement of a complete response may have abrogated the prognostic significance of cell of origin by immunohistochemistry.
Selpercatinib, a highly selective potent and brain-penetrant RET inhibitor, was shown to have efficacy in patients with advanced RET fusion–positive non–small-cell lung cancer (NSCLC) in a nonrandomized phase 1–2 study. Download a PDF of the Research Summary. In a randomized phase 3 trial, we evaluated the efficacy and safety of first-line selpercatinib as compared with control treatment that consisted of platinum-based chemotherapy with or without pembrolizumab at the investigator's discretion. The primary end point was progression-free survival assessed by blinded independent central review in both the intention-to-treat–pembrolizumab population (i.e., patients whose physicians had planned to treat them with pembrolizumab in the event that they were assigned to the control group) and the overall intention-to-treat population. Crossover from the control group to the selpercatinib group was allowed if disease progression as assessed by blinded independent central review occurred during receipt of control treatment. In total, 212 patients underwent randomization in the intention-to-treat–pembrolizumab population. At the time of the preplanned interim efficacy analysis, median progression-free survival was 24.8 months (95% confidence interval [CI], 16.9 to not estimable) with selpercatinib and 11.2 months (95% CI, 8.8 to 16.8) with control treatment (hazard ratio for progression or death, 0.46; 95% CI, 0.31 to 0.70; P<0.001). The percentage of patients with an objective response was 84% (95% CI, 76 to 90) with selpercatinib and 65% (95% CI, 54 to 75) with control treatment. The cause-specific hazard ratio for the time to progression affecting the central nervous system was 0.28 (95% CI, 0.12 to 0.68). Efficacy results in the overall intention-to-treat population (261 patients) were similar to those in the intention-to-treat–pembrolizumab population. The adverse events that occurred with selpercatinib and control treatment were consistent with those previously reported. Treatment with selpercatinib led to significantly longer progression-free survival than platinum-based chemotherapy with or without pembrolizumab among patients with advanced RET fusion–positive NSCLC. (Funded by Eli Lilly and others; ClinicalTrials.gov number, NCT04194944.) QUICK TAKE VIDEO SUMMARYFirst-Line Selpercatinib in RET Fusion–Positive NSCLC 01:52
2516 Background: Skeletal muscle wasting (cachexia) is a prevalent and not readily managed condition in advanced cancer patients. LY2495655 is a humanized monoclonal antibody to myostatin, which has demonstrated positive effects on cachexia measures in animal models. We present phase I trial data on use of LY2495655 in healthy volunteers (Study 1) and interim data from an ongoing phase I study in patients with advanced cancer (Study 2). Methods: Study 1 was a randomized, placebo-controlled, blinded, single-dose, parallel, dose-escalation study evaluating the safety and tolerability of IV or SC LY2495655 (0.7 mg-700 mg). Study 2 is an ongoing nonrandomized, open-label study evaluating the safety and pharmacokinetics (PKs) of LY2495655 in patients with advanced cancer not receiving chemotherapy. Dose cohorts (2 mg-700 mg, ≥3 patients per cohort) were to be treated until the maximum tolerated dose (MTD) was met, or the highest dose (700 mg) cohort was completed. Final locked data from Study 1 and interim data from the dose escalation phase of Study 2 were used in the analyses. Results: In Study 1, 64 healthy volunteers were enrolled (48 LY2495655, 16 placebo). In Study 2, 22 patients had received treatment with LY2495655 at the time of the analysis. In both studies, all doses of LY2495655 were well tolerated (no DLTs were observed and MTD was not reached), and nonlinear PKs were observed (most evident in lower dose levels). In Study 1, thigh muscle volume generally increased with LY2495655. In Study 2, increased muscle volume was observed only at 21-mg and 70-mg doses. Consistent increases in hand grip strength and improvements in functional tests were observed at doses ≥21 mg. Conclusions: There were no unusual safety concerns in healthy subjects or cancer patients. PK results were consistent between the 2 studies. Increases in muscle volume were observed in both studies, with concomitant improvement in functional measures. However, there is no clear trend in dose-dependent efficacy, possibly due to extremely small sample sizes and patient heterogeneity. Enrollment in Study 2 continues with dose expansion cohorts. A Phase 2 study is ongoing in pancreatic cancer patients.
5009 Background: Preclinical and phase 1 results suggest PI3K/mTOR pathway inhibition may enhance androgen receptor inhibition. We report the results of a double-blind, placebo-controlled, randomized Phase 1b/2 study of ENZ±LY (a dual PI3K/mTOR inhibitor) in pts with mCRPC who progressed on abiraterone. Methods: Phase 1b pts received single-agent LY 200 mg twice daily (BID) for 1 wk prior to starting LY+ENZ. Phase 2 pts were randomized 1:1 to 160 mg daily ENZ with PL or 200 mg BID LY on a 28-d cycle. The primary objective was progression-free survival (PFS: serological, radiographic [rPFS], or death) by PCWG2 criteria. Secondary objectives were rPFS, safety, decline in PSA, and PK. Exploratory biomarker analyses included outcomes by presence of androgen receptor variant 7 (AR-V7). 92 primary PFS events were needed for the study to have at least 80% power at one-sided alpha=0.20. Results: LY+ENZ was tolerable during Phase 1b with 1 dose limiting toxicity observed in 13 enrolled pts. Mean LY exposures remained in an efficacious range despite a 30% average decrease when combined with ENZ. In Phase 2, 129 pts were randomized to LY+ENZ (N=65) and PL+ENZ (N=64) (Table). Median PCWG2-PFS was 3.7 mos (LY+ENZ) vs 2.9 mos (PL+ENZ) (HR 0.66, 95% CI 0.43, 0.99; p-value 0.0208). Conclusions: Combination LY+ENZ had a clinically manageable safety profile. The primary end-point of PCWG2-PFS was met and is supported by a clinically meaningful delay in rPFS in AR-V7 negative pts. The biomarker data provide important insights to inform future development strategies. Clinical trial information: NCT02407054. [Table: see text]
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. Sci. 1992; 17: 414-417Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 14Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4224) Google Scholar, 15Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2362) Google Scholar, 16Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4035) Google Scholar, 17Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 18Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar) including integrin-mediated cell spreading and migration (19Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 277-290Crossref PubMed Google Scholar, 20Klemke R.L. Yebra M. Bayna E.M. Cheresh D.A. J. Cell Biol. 1994; 127: 859-866Crossref PubMed Scopus (248) Google Scholar, 21Yebra M. Filardo E.J. Bayna E.M. Kawahara E. Becker J.C. Cheresh D.A. Mol. Biol. Cell. 1995; 6: 841-850Crossref PubMed Scopus (77) Google Scholar, 22Timar J. Trikha M. Szekeres K. Bazaz R. Tovari J. Silletti S. Raz A. Honn K.V. Cancer Res. 1996; 56: 1902-1908PubMed Google Scholar). To date, 11 isoenzymes of the serine/threonine kinase PKC have been identified and classified into three subgroups based on structure and cofactor regulation: conventional PKC, novel PKC, and atypical PKC (15Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2362) Google Scholar, 18Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar). The conventional PKC isoforms participate in the inside-out signaling activation of cell adhesion mediated by β1, β2, and β3 integrins and are also required for cell spreading (23Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar, 24Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1286) Google Scholar, 25Shimizu Y. Van Seventer G.A. Horgan K.J. Shaw S. Nature. 1990; 345: 250-253Crossref PubMed Scopus (540) Google Scholar). Moreover, PKCα associates with β1 integrins, thereby regulating cell trafficking (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,27Ng T. Parsons M. Hughes W.E. Monypenny J. Zicha D. Gautreau A. Arpin M. Gschmeissner S. Verveer P.J. Bastiaens P.I. Parker P.J. EMBO J. 2001; 20: 2723-2741Crossref PubMed Scopus (248) Google Scholar). In the present study, we describe the close interrelationship between integrin and growth factor-induced signaling pathways in MM. We identify PKCα as the primary PKC isozyme involved in VEGF-induced MM cell migration. 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&D Systems (Minneapolis, MN). Human plasma fibronectin was obtained from Invitrogen. β1 integrin-specific mAb was purified from P4C10 ascites (Chemicon, Temecula, CA). PKC isoforms were purchased from Transduction Laboratories. The goat polyclonal Ab raised against the carboxyl terminus of PYK2, the mouse mAb raised against full-length β1 integrin (4B7R), and the rabbit polyclonal Ab directed against amino acids within the extracellular domain of Flt-1 were purchased from Santa Cruz. Anti-phosphotyrosine 4G10 antibody was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA). The human MM cell line MM.1S (dexamethasone-sensitive) (28Moalli P.A. Pillay S. Weiner D. Leikin R. Rosen S.T. Blood. 1992; 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% FBS overnight and then for 3 h without FBS prior to stimulation with indicated VEGF concentrations (recombinant human VEGF, R&D Systems) or 50 nm/300 nm PMA for 20–30 min at 37 °C. Cells were washed three times with phosphate-buffered saline and lysed with either lysis buffer (10 mm Tris, 50 mm NaCl, Na-pyrophosphate, 1% Triton, 1 mmsodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and protein inhibitor mixture; Roche Molecular Biochemicals) or radioimmune precipitation assay lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% v/v Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture). Insoluble material was removed by centrifugation (15,000 rpm for 30 min at 4 °C). Immunocomplexes were collected following overnight incubation at 4 °C with 10–20 μl of 100% protein A-Sepharose CL-4B beads (Amersham Biosciences, Inc.). For Western blotting, cell lysates (30–100 μg/lane) or immunoprecipitates (500 μg–1.5 mg total proteins) were separated by 8 or 10% SDS-PAGE prior to electrophoretic transfer onto HybondTM-C super nitrocellulose membranes (Amersham Biosciences, Inc.). After blocking with 5% nonfat milk in phosphate-buffered saline-Tween 20 buffer at room temperature for 1 h, membranes were sequentially blotted with the indicated specific primary Abs and then with horseradish peroxidase-conjugated secondary mouse, rabbit, or goat Abs and were developed using chemiluminescence (Amersham Biosciences, Inc.). After washing three times with phosphate-buffered saline, cells were transferred into 200 μl of hypotonic lysis buffer (HLB: 10 mm Tris-HCl, 1 mm EDTA, 1 mm sodium vanadate, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture) and incubated for 20 min on ice. The cells were then lysed by 80 strokes in a Dounce homogenizer and subjected to centrifugation at 1500 × g to pellet nuclei and unbroken cells followed by centrifugation of the supernatant at 100,000 × g for 20 min. The supernatant was collected (S100 fraction), and the pellet was resuspended in 70 μl of HLB containing 0.1% Triton X (P100 fraction). PKCα activity was measured with a PKC assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. The phosphotransferase activity of PKCα was quantitated in a scintillation counter (Beckman LS 6500, multi-purpose scintillation counter) measuring the amount of the γ-phosphate of [γ-32P]ATP incorporated in a specific PKC substrate peptide (QKRPSQRSKYL) bound to P81 phosphocellulose paper. Endogenous phosphorylation of proteins in the sample was determined by substituting the assay dilution buffer for the substrate mixture. To assure that equal amounts of PKCα were used in the assay, immunoprecipitates were denaturated, eluted, separated by 10% SDS-PAGE, electrophoretically transferred, and immunoblotted with PKCα. PI 3-kinase assays were performed as described previously (29Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. Cell. 1989; 57: 167-175Abstract Full Text PDF PubMed Scopus (681) Google Scholar) in a total volume of 50 μl. The radioactivity was visualized and quantitated on a PhosphorImager (Amersham Biosciences, Inc.). These were performed using the VybrantTM cell adhesion assay kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Briefly, after an 18-h starvation in RPMI/2% FBS, MM.1S cells (4 × 106 cells/ml) were harvested and labeled with calcein-acetoxymethyl ester (5 μm per cell loading) for 30 min. After washing with prewarmed (37 °C) RPMI 1640 (without serum), the cells were preincubated with or without blocking β1 integrin neutralizing mAb, bisindolylmaleimide I (BIM I, Calbiochem), or LY294002 (Calbiochem), respectively, and then stimulated with VEGF. The cell suspensions were immediately added to fibronectin- or polylysine (1 μg/ml)-coated or non-coated wells. After 90–120 min, nonadherent calcein-labeled cells were removed by gently washing twice with RPMI 1640 by inversion of the plates. Adherent cells were quantitated in a fluorescence multi-well plate reader (Molecular Devices, Sunnyvale, CA) and examined microscopically. All experiments were done in triplicate. Cell migration was assayed as described previously (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, 30Kundra V. Anand-Apte B. Feig L.A. Zetter B.R. J. Cell Biol. 1995; 130: 725-731Crossref PubMed Scopus (103) Google Scholar, 31Kuzuya M. Kinsella J.L. Exp. Cell Res. 1994; 215: 310-318Crossref PubMed Scopus (43) Google Scholar). After 2–5 h, cells that had migrated into the lower compartment of a Boyden-modified chamber were counted using a Coulter counter ZBII (Beckman Coulter). MM.1S cells were starved in RPMI medium containing 2% FBS for 16 h and plated to uncoated or fibronectin-coated tissue culture plates (35 × 10-mm plates, BD PharMingen), respectively, in the presence or absence of VEGF (100 ng/ml). For image capturing, an Olympus IX70 inverted microscope (Olympus, Lake Success, NY) with Hoffman optics (×10/×20/×40) equipped with a temperature- (Therm-Omega-Tech, Warmington, PA) and CO2 (5%)-controlled chamber was connected to an Optronics Engineering DEI-750 3CCD digital video camera (Optronics, Galeta, CA). Animation and export to a Quick Time movie were performed using the QED Camera Standalone 145 software at 2-min intervals. Images were analyzed with the NIH Image 1.62 software. To generate migration tracks, the position of the centroid of individual cells on each image were marked. The migratory speed was calculated based on the sum of distances divided by the time of observation. Migration of at least 15 cells was analyzed for each experimental condition. Statistical significance of differences observed in VEGF-treated versus control cultures was determined using an unpaired Student's t test. The minimal level of significance was p < 0.05. Previously 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 define and characterize the interrelationship of VEGF- and integrin-signaling in activating PKC that ultimately leads to MM cell migration. Several PKC isoforms, including PKCα (32Platet N. Prevostel C. Derocq D. Joubert D. Rochefort H. Garcia M. Int. J. Cancer. 1998; 75: 750-756Crossref PubMed Scopus (74) Google Scholar), PKCδ (33Kiley S.C. Clark K.J. Goodnough M. Welch D.R. Jaken S. Cancer Res. 1999; 59: 3230-3238PubMed Google Scholar), and PKCθ (34Tang S. Morgan K.G. Parker C. Ware J.A. J. Biol. Chem. 1997; 272: 28704-28711Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), have been implicated in a cell migratory phenotype. Additionally, overexpression of PKCα was shown to enhance cell motility/invasiveness of breast 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 shown previously that MM cell migration is PKC-dependent because it can be selectively inhibited by the PKC inhibitor BIM 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). As a first step to identify the class of PKC mediating VEGF-induced migration in MM cells, we examined the expression of the PKC isoforms in MM cell lines and patient cells (Fig. 1). Immunoblot analysis revealed that PKCα, PKCγ, PKCε, and PKCζ are significantly expressed in all human MM cell lines and MM patient cells investigated, in contrast to PKCβ (low expression), PKCδ (variable expression), and PKCθ (no expression, data not shown). As in our previous studies we chose 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 representative model system for this study. Because the activation of PKC occurs concomitantly with recruitment to the plasma membrane, we next performed immunoblotting of cell fractions with isoform-specific Abs against PKCα, PKCγ, PKCε, and PKCζ to delineate the intracellular distribution after VEGF stimulation (Fig.2). In non-treated MM cells all PKC isoforms were detected primarily in the cytosolic fraction. After VEGF stimulation of cells seeded on fibronectin, PKCα translocated into the membrane fraction after 30 min (Fig. 2 a), whereas no significant changes in distribution of PKC isoforms as PKCγ, PKCε, and PKCζ were observed. The activation of PKCα after VEGF treatment of cells attached to fibronectin was further supported by a 2-fold increase in PKCα-IP kinase activity; PKCα-IP kinase activation by PMA served as a positive control (Fig. 2 b). Although a cAMP-dependent protein kinase/calmodulin kinase (PKA/CaMK) inhibitor mixture was used, this assay (Upstate Biotechnology) may not exclude the phosphorylation of a specific substrate peptide (QKRPSQRSKYL) by unknown PKCα-coprecipitated kinases. Neither plating of cells on fibronectin alone nor stimulation of suspended cells with VEGF alone significantly changed the subcellular distribution of PKCα (Fig. 2, c andd). In contrast, treatment with PMA, a major PKC activator, induced translocation of responsive PKC (PKCα, PKCγ, and PKCε) into the detergent-soluble membrane fraction as expected (Fig.2 d) (35Kraft A.S. Anderson W.B. Nature. 1983; 301: 621-623Crossref PubMed Scopus (1001) Google Scholar, 36Kraft A.S. Anderson W.B. Cooper H.L. Sando J.J. J. Biol. Chem. 1982; 257: 13193-13196Abstract Full Text PDF PubMed Google Scholar) Laminin, the microfibrillar collagen type VI, and fibronectin bind 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 include 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, 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), which mediate adherence to both the ECM and BM stromal cells. In human umbilical vein endothelial cells, VEGF stimulates β1 integrins via inside-out signaling, leading to significantly increased motility (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). Because migration is a dynamic process of cell adhesion formation and release, we next investigated whether VEGF can modulate β1integrin-mediated MM cell adhesion. As shown previously, MM cells spontaneously adhered to fibronectin, and this adhesion was increased upon stimulation with VEGF (Fig.3 a). Maximal increments of VEGF-mediated cell adhesion were observed at fibronectin concentrations of 25–30 μg/ml, whereas adhesion decreased to baseline levels at higher fibronectin concentrations (Fig. 3 b). Notably, VEGF-mediated increments in adhesion were time-dependent, with maximal binding observed after 75–90 min of VEGF treatment and decreasing after 120 min (Fig. 3 c). We next examined whether the increment of adhesion observed at 90 min of VEGF stimulation was PKC- and β1integrin-dependent. As shown in Fig. 3,d–e, incubation with the PKC inhibitor BIM I (as well as with the β1-neutralizing mAb) blocked dose-dependent VEGF-induced cell adhesion to fibronectin. This involvement of PKC in MM cell adhesion to fibronectin was further confirmed by a dose-dependent increment of adhesion triggered by PMA stimulation (Fig. 3 d) similar to that induced by VEGF. Taken together, our results show that VEGF transiently enhances MM cell adhesion to fibronectin, dependent on both PKC and β1 integrin activity. We next sought to determine whether β1integrin modulates VEGF-mediated MM cell migration on fibronectin. As seen in Fig. 3 f, β1 integrin neutralizing mAb (but not irrelevant IgG) mediated dose-dependent inhibition of VEGF-triggered MM cell migration in a Boyden-modified microchemotaxis chamber. These data confirm that β1integrin (CD29) is the integrin primarily associated with VEGF-triggered MM cell migration on fibronectin. The control mechanisms leading to the various stages of the integrin receptor life cycle are largely unknown. Propagation of cell movement is thought to be regulated by the distribution and redistribution of integrins through surface diffusion, internalization, clustering at the leading front, and ripping release from the cell rear (37Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3274) Google Scholar). In mammary epithelial cells Nget al. (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 interacts with activated β1 integrin, which regulates its exocytosis to the plasma membrane; moreover, catalytically active PKCα is responsible for β1 integrin internalization through a Ca2+- and PI 3-kinase-dependent, dynamin I-controlled endocytic pathway. PKCα induced up-regulation of the integrin-dependent cell migration of these cells, which was blocked under conditions that prevented the internalization of the receptor complex. We therefore next determined whether PKCα and β1 integrin are associated in MM cells. Constitutive complex formation between these two proteins was demonstrated by co-immunoprecipitation (Fig.4 a). Upon activation and translocation, conventional PKCs associate with proteins of the transmembrane-4 superfamily (TM4SF or tetraspans) linking PKC to several subsets of integrins (38Hemler M.E. Curr. Opin. 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In addition to influencing cell motility via controlling β1 integrin trafficking, catalytically active PKC also regulates other components of the focal complexes, including the small GTPases (Cdc42, Rho, Rac) and/or actin cytoskeleton-binding proteins. Calcium-dependent tyrosine kinase (CADTK), also known as proline-rich tyrosine kinase 2 (PYK2), calcium-dependent tyrosine-kinase β (CAKβ), and related adhesion focal tyrosine kinase (RAFTK), is a cytoplasmic tyrosine kinase homologous to focal adhesion kinase (FAK). Like FAK, CADTK is a platform kinase site for the coalescence of signaling and adaptor molecules, thereby facilitating the transmission of surface signals to the cytoskeleton and signaling pathways associated with cell growth, apoptosis, and migration. A number of studies have demonstrated tyrosine phosphorylation of CADTK in cells of hematopoietic origin (e.g. T and B cells, monocytes, natural killer cells, granulocytes, bone marrow progenitors, mast cells, megakaryocytes, and platelets). Stimuli that activate CADTK in these cells are associated with cell motility or at least cytoskeletal rearrangement (e.g. CADTK activation is required for cytoskeletal reorganization and monocyte motility) (42Watson J.M. Harding T.W. Golubovskaya V. Morris J.S. Hunter D. Li X. Haskill J.S. Earp H.S. J. Biol. Chem. 2001; 276: 3536-3542Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In MM.1S cells, we have shown previously that CADTK is activated upon dexamethasone treatment, suggesting its role in dexamethasone-induced apoptosis (47Chauhan D. Hideshima T. Pandey P. Treon S. Teoh G. Raje N. Rosen S. Krett N. Husson H. Avraham S. Kharbanda S. Anderson K.C. Oncogene. 1999; 18: 6733-6740Crossref PubMed Scopus (103) Google Scholar). 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