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Protein Kinase Cϵ Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis

2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês

10.1074/jbc.m705839200

ISSN

1083-351X

Autores

Lynn Y.L. Huang, Hung-Chi Cheng, Richard Isom, Chia-Sui Chen, Roy A. Levine, Bendicht U. Pauli,

Tópico(s)

HER2/EGFR in Cancer Research

Resumo

Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cϵ (PKCϵ), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCϵ protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCϵ and dominant-negative PKCϵ constructs (e.g. RD-PKCϵ). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCϵ impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis. Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cϵ (PKCϵ), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCϵ protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCϵ and dominant-negative PKCϵ constructs (e.g. RD-PKCϵ). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCϵ impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis. Fibronectin (Fn) 2The abbreviations used are: FnfibronectinpFnplasma FnPKCprotein kinase CcPKCconventional PKCnPKCnovel PKC isoformcFncellular FnPKCprotein kinase CRDregulatory domainPEphycoerythrinPLCphospholipase CBIM Ibisindolylmaleimide Iwtwild typentnucleotidesGFPgreen fluorescent proteinFACSfluorescence-activated cell sortingsi-small interfering-EoEend-over-endFBSfetal bovine serumFFSFn-free FBSPBSphosphate-buffered salineDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPPdipeptidylpeptidasePMAphorbol 12-myristate 13-acetateHBDDE2,2′ 3,3′ 4,4′-hexahydroxyl-1,1′-biphenyl-6,6′ dimethanol dimethyl ether. 2The abbreviations used are: FnfibronectinpFnplasma FnPKCprotein kinase CcPKCconventional PKCnPKCnovel PKC isoformcFncellular FnPKCprotein kinase CRDregulatory domainPEphycoerythrinPLCphospholipase CBIM Ibisindolylmaleimide Iwtwild typentnucleotidesGFPgreen fluorescent proteinFACSfluorescence-activated cell sortingsi-small interfering-EoEend-over-endFBSfetal bovine serumFFSFn-free FBSPBSphosphate-buffered salineDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminDPPdipeptidylpeptidasePMAphorbol 12-myristate 13-acetateHBDDE2,2′ 3,3′ 4,4′-hexahydroxyl-1,1′-biphenyl-6,6′ dimethanol dimethyl ether. is a "pro-metastatic" gene that is overexpressed in several malignancies (1Zhang L. Zhou W. Velculescu V.E. Kern S.E. Hruban R.H. Hamilton S.R. Vogelstein B. Kinzler K.W. Science. 1997; 276: 1268-1272Crossref PubMed Scopus (1222) Google Scholar, 2Bittner M. Meltzer P. Chen Y. Jiang Y. Seftor E. Hendrix M. Radmacher M. Simon R. Yakhini Z. Ben-Dor A. Sampas N. Dougherty E. Wang E. Marincola F. Gooden C. Lueders J. Glatfelter A. Pollock P. Carpten J. Gillanders E. Leja D. Dietrich K. Beaudry C. Berens M. Alberts D. Sondak V. Nature. 2000; 406: 536-540Crossref PubMed Scopus (1697) Google Scholar, 3Al Moustafa A.E. Alaoui-Jamali M.A. Batist G. Hernandez-Perez M. Serruya C. Alpert L. Black M.J. Sladek R. Foulkes W.D. Oncogene. 2002; 21: 2634-2640Crossref PubMed Scopus (189) Google Scholar, 4Jiang Y. Harlocker S.L. Molesh D.A. Dillon D.C. Stolk J.A. Houghton R.L. Repasky E.A. Badaro R. Reed S.G. Xu J. Oncogene. 2002; 21: 2270-2282Crossref PubMed Scopus (71) Google Scholar, 5Amatschek S. Koenig U. Auer H. Steinlein P. Pacher M. Gruenfelder A. Dekan G. Vogl S. Kubista E. Heider K.H. Stratowa C. Schreiber M. Sommergruber W. Cancer Res. 2004; 64: 844-856Crossref PubMed Scopus (202) Google Scholar, 6Hao X. Sun B. Hu L. S̈hdesmS̈ki L.H. Dumire V. Feng Y. Zhang S.W. Wang H. Wu C. Wang H. Fuller G.N. Symmans W.F. Shumulevich I. Zhang W. Cancer. 2004; 100: 1110-1122Crossref PubMed Scopus (153) Google Scholar, 7Zucchi I. Mento E. Kuznetsov V.A. Scotti M. Valsecchi V. Simionati B. Vicinanza E. Valle G. Pilotti S. Reinbold R. Vezzoni P. Albertini A. Dulbecco R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 18147-18152Crossref PubMed Scopus (93) Google Scholar) and, most prominently, in cancer cell lines selected for enhanced lung colonization (8Korach S. Poupon M.F. Du Villard J.A. Becker M. Cancer Res. 1986; 46: 3624-3629PubMed Google Scholar, 9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Nature. 2000; 406: 532-535Crossref PubMed Scopus (1303) Google Scholar, 11Bandyopadhyay A. Elkahloun A. Baysa S.J. Wang L. Sun L.Z. Cancer Biol. Ther. 2005; 4: 168-174Crossref PubMed Google Scholar). This pro-metastatic role of Fn is multifaceted, affecting several steps of the metastatic cascade by modulating cell adhesion, motility/invasion, cell cycle progression, and cell survival (for review, see Refs. 12Hynes R.O. Yamada K.M. J. Cell Biol. 1982; 95: 369-377Crossref PubMed Scopus (939) Google Scholar, 13Humphries M.J. Obara M. Olden K. Yamada K.M. Cancer Investig. 1989; 7: 373-393Crossref PubMed Scopus (97) Google Scholar, 14Ruoslahti E. Adv. Cancer Res. 1999; 67: 1-20Google Scholar, 15Danen E.H. Yamada K.M. J. Cell. Physiol. 2001; 189: 1-13Crossref PubMed Scopus (394) Google Scholar, 16Labat-Robert J. Semin. Cancer Biol. 2002; 12: 187-195Crossref PubMed Scopus (98) Google Scholar). By analyzing cancer cells that had entered the blood circulation from malignant breast cancers implanted into the mammary fat pad of rats or mice, we discovered that blood-borne tumor cells were decorated with a unique, multiglobular coat of polymeric Fn (polyFn) (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). PolyFn aggregates appeared to arise from focal accumulations of endogenous, cell surface-immobilized ("linearized") Fn, which served as scaffolds for further Fn-self-assembly from Fn recruited from blood plasma (pFn) (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Aggregates typically became increasingly deoxycholate-insoluble as they increased in size with time of incubation of suspended cancer cells in serum-containing medium in vitro. Biochemically, aggregates impressed as prominent, insoluble (covalently bonded) Fn polymers sitting on top of the stacks of SDS-polyacrylamide gels and, immunocytochemically, as large globules randomly dispersed over the entire cancer cell surface (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This "cluster arrangement" of polyFn was shown to have the following functional implications. First, the conversion of Fn from the globular state of soluble Fn to the linearized state of insoluble, surface-associated Fn aggregates is associated with exposure of a novel, cryptic binding domain for the lung endothelial cell address in dipeptidylpeptidase IV (DPP IV) (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Johnson R.C. Augustin-Voss H.G. Zhu D. Pauli B.U. Cancer Res. 1991; 51: 394-399PubMed Google Scholar, 19Johnson R.C. Zhu D. Augustin-Voss H.G. Pauli B.U. J. Cell Biol. 1993; 121: 1423-1432Crossref PubMed Scopus (88) Google Scholar, 20Abdel-Ghany M. Cheng H. Levine R.A. Pauli B.U. Invasion Metastasis. 1998; 18: 35-43Crossref PubMed Scopus (31) Google Scholar, 21Cheng H.C. Abdel-Ghany M. Zhang S. Pauli B.U. Clin. Exp. Metastasis. 1999; 17: 609-615Crossref PubMed Scopus (30) Google Scholar). This DPP IV binding domain is present as a consensus motif in each of the 13th, 14th, and 15th type III repeats of Fn (17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Second, the DPP IV binding specificity for linearized (polymeric), but not for globular (soluble) pFn, allows tumor cell adhesion to endothelial DPP IV in the presence of high pFn concentrations (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Third, the large Fn aggregates on cancer cell surfaces allow multiple binding interactions with endothelial DPP IV molecules, thereby generating adhesion strengths between cancer cells and endothelial cells that are able to withstand the rigors of hemodynamic shear stresses (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The importance of the Fn/DPP IV-docking mechanism is substantiated by our discovery that synthetic peptides directed against the DPP IV binding domain in the 13th, 14th, or 15th type III repeats of Fn as well as a polypeptide encompassing the bulk of the extracellular domain of DPP IV dramatically impeded pulmonary metastasis in a rat breast cancer model (19Johnson R.C. Zhu D. Augustin-Voss H.G. Pauli B.U. J. Cell Biol. 1993; 121: 1423-1432Crossref PubMed Scopus (88) Google Scholar, 20Abdel-Ghany M. Cheng H. Levine R.A. Pauli B.U. Invasion Metastasis. 1998; 18: 35-43Crossref PubMed Scopus (31) Google Scholar). These data are consistent with our finding that colonization of the lungs was greatly diminished in Fischer 344/CRJ rats, in which DPP IV is mutated causing a significantly decreased DPP IV protein expression in pulmonary endothelia (21Cheng H.C. Abdel-Ghany M. Zhang S. Pauli B.U. Clin. Exp. Metastasis. 1999; 17: 609-615Crossref PubMed Scopus (30) Google Scholar) as well as in DPP IV-/- mice (19Johnson R.C. Zhu D. Augustin-Voss H.G. Pauli B.U. J. Cell Biol. 1993; 121: 1423-1432Crossref PubMed Scopus (88) Google Scholar). DPP IV-/- mice injected with lung-metastatic cancer cells lived significantly longer than their wild-type counterparts, an outcome granted by the formation of significantly fewer and smaller lung colonies. 3H. C. Cheng, unpublished data. 3H. C. Cheng, unpublished data.Although we have firmly established a critical dependence between the ability of the cancer cells to assemble an insoluble, globular polyFn-surface coat and lung colonization in an experimental metastasis model (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), we still do not know whether blood-borne cancer cells use their own cellular Fn (cFn) or rely on ubiquitous pFn to assemble their polyFn surface coat, how cancer cells regulate the polyFn build-up, and how Fn cell surface deposits are transformed into covalently bonded, insoluble aggregates (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To answer some of these questions we examined polyFn genesis in lung-metastatic MTF7L rat breast cancer cells subjected to end-over-end (EoE) suspension culture in serum- or pFn-containing medium, which together induce and augment the build-up of a polyFn surface coat similar to that observed on tumor cells that have entered the blood circulation (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The data presented here show that assembly of the prometastatic, multiglobular polyFn surface coat on MTF7L breast cancer cells depends upon the synthesis and secretion of endogenous, cellular Fn (Fn1: EDA+EDB+IIICS120-Fn) and is regulated by membrane-translocation and Ser/Thr phosphorylation of protein kinase Cϵ (PKCϵ). Inhibitors of protein synthesis, protein secretion, and novel PKC isoforms (nPKCs) as well as transfection of MTF7L cells with the PKCϵ regulatory domain (RD) or PKCϵ siRNA species all substantially decrease the ability of the cancer cells to assemble a polyFn surface coat. Functionally, inability to assemble polyFn is associated with failure to colonize the lungs. Together, our studies provide novel insights of the regulation of Fn in suspended (blood-borne) breast cancer cells and provide a renewed appreciation for the previously recognized role of Fn in metastasis (1Zhang L. Zhou W. Velculescu V.E. Kern S.E. Hruban R.H. Hamilton S.R. Vogelstein B. Kinzler K.W. Science. 1997; 276: 1268-1272Crossref PubMed Scopus (1222) Google Scholar, 2Bittner M. Meltzer P. Chen Y. Jiang Y. Seftor E. Hendrix M. Radmacher M. Simon R. Yakhini Z. Ben-Dor A. Sampas N. Dougherty E. Wang E. Marincola F. Gooden C. Lueders J. Glatfelter A. Pollock P. Carpten J. Gillanders E. Leja D. Dietrich K. Beaudry C. Berens M. Alberts D. Sondak V. Nature. 2000; 406: 536-540Crossref PubMed Scopus (1697) Google Scholar, 3Al Moustafa A.E. Alaoui-Jamali M.A. Batist G. Hernandez-Perez M. Serruya C. Alpert L. Black M.J. Sladek R. Foulkes W.D. Oncogene. 2002; 21: 2634-2640Crossref PubMed Scopus (189) Google Scholar, 4Jiang Y. Harlocker S.L. Molesh D.A. Dillon D.C. Stolk J.A. Houghton R.L. Repasky E.A. Badaro R. Reed S.G. Xu J. Oncogene. 2002; 21: 2270-2282Crossref PubMed Scopus (71) Google Scholar, 5Amatschek S. Koenig U. Auer H. Steinlein P. Pacher M. Gruenfelder A. Dekan G. Vogl S. Kubista E. Heider K.H. Stratowa C. Schreiber M. Sommergruber W. Cancer Res. 2004; 64: 844-856Crossref PubMed Scopus (202) Google Scholar, 6Hao X. Sun B. Hu L. S̈hdesmS̈ki L.H. Dumire V. Feng Y. Zhang S.W. Wang H. Wu C. Wang H. Fuller G.N. Symmans W.F. Shumulevich I. Zhang W. Cancer. 2004; 100: 1110-1122Crossref PubMed Scopus (153) Google Scholar, 7Zucchi I. Mento E. Kuznetsov V.A. Scotti M. Valsecchi V. Simionati B. Vicinanza E. Valle G. Pilotti S. Reinbold R. Vezzoni P. Albertini A. Dulbecco R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 18147-18152Crossref PubMed Scopus (93) Google Scholar).EXPERIMENTAL PROCEDURESAntibodies and Reagents—Rabbit anti-PKCδ, -PKCϵ, -PKCη, -PKCθ, -PKCζ, and anti-hemagglutinin tag polyclonal antibodies and mouse anti-Fn (raised against a region in the human Fn-EDA domain; human- mouse-, and rat-specific) monoclonal antibody (anti-Fn[EDA]) were from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit anti-Fn polyclonal antibodies that recognize pFn and cFn from both bovine and rat (anti-Fn[pan]; does not cross-react with fibrinogen, vitronectin, laminin, collagen type IV) was from Sigma, rabbit anti-PKCϵ polyclonal antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY), mouse anti-PKCϵ used for immunoprecipitation was from BD Biosciences, phycoerythrin (PE)-conjugated donkey anti-rabbit, PE-conjugated goat anti-mouse, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit, and HRP-conjugated goat anti-mouse antibodies were from Jackson ImmunoResearch (West Grove, PA), and rabbit anti-PKCα, -vinculin, and -actin were from Dr. Guan (University of Michigan). Pertussis toxin (Gαi inhibitor), PD98059 (MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 inhibitor), SU6656 and PP2 (Src family kinase inhibitors), wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors), JAKI (Janus family kinase 1/2 inhibitor), Y27632 (ROCK1/2 inhibitor), U73122 (inhibitor of phosphatidylinositol-specific phospholipase C (PLC)), the PKC inhibitors calphostin C, Gö6976, HBDDE, bisindolylmaleimide I (BIM I), Gö6983, BIM XI (Ro-32-0432), rottlerin, brefeldin A, monensin, and cycloheximide were from EMD Chemicals (San Diego, CA). Fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Woodland, CA). Fn-free FBS (FFS) was generated by successive gelatin- and anti-Fn antibody affinity chromatography (22Hayman E.G. Ruoslahti E. J. Cell Biol. 1979; 83: 255-259Crossref PubMed Scopus (161) Google Scholar). All other chemicals and reagents were from Sigma.Cell Cultures—MTF7L cells were derived from a lung metastasis generated by tail-vein injection of MTF7 breast cancer cells (obtained from Dr. D. R. Welch, University of Alabama at Birmingham, Birmingham, AL) into Fischer 344 rats. At an intravenous inoculation dose of 2 × 105 cells per rat, MTF7L cells consistently produce in excess of 400 lung colonies. Cells were grown in culture in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated FBS. For EoE suspension culture, MTF7L cells were grown to 80-90% confluence, then removed from the growth surface by trypsinization (0.25% trypsin, 0.02% EDTA in phosphate-buffered saline (PBS) for 10 min at 37 °C), washed twice in DMEM containing 10% FBS, and subjected to EoE suspension culture for 1 h (or as indicated) in 2-ml centrifuge tubes in DMEM plus 20% FBS at a concentration of 5 × 106 cells/ml (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Abdel-Ghany M. Cheng H. Levine R.A. Pauli B.U. Invasion Metastasis. 1998; 18: 35-43Crossref PubMed Scopus (31) Google Scholar). Tumor cells were used for all experiments within 10 passages from frozen stocks that were tested for metastatic performance immediately before freezing.For metabolic labeling, MTF7L cells in logarithmic growth phase were labeled with [35S]methionine (0.33 mCi/3 ml) in methionine-free DMEM (both from MP Biomedicals, Solon, OH) containing 20 μm methionine and 10% dialyzed, Fn-free FBS as previously described (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). For 32P labeling, cells were serum-starved overnight, then incubated for 4 h in phosphate-free DMEM containing 100 μCi/ml [32P]orthophosphate (ICN Biochemicals, Irvine, CA) and washed in 3 changes of PBS. Labeled cells were subjected to EoE suspension culture as describe above and immediately processed for biochemical analyses.Plasmid Constructs, Transfection, and Selection—The constructs wtPKCϵ, RD-PKCϵ, and RD-PKCη cloned into pEGFP-N1 were obtained from Dr. C. Larsson (Lund University, Malmö, Sweden) (23Zeidman R. Ls̈fgren B. Pahlman S. Larsson C. J. Cell Biol. 1999; 145: 713-726Crossref PubMed Scopus (110) Google Scholar), and wtPKCδ and RD-PKDδ were cloned into pcDNA3.1 from Dr. D. Mayer (Deutsches Krebsforschungszentrum, Heidelberg, Germany) (24De Servi B. Hermani A. Medunjanin S. Mayer D. Oncogene. 2005; 24: 4946-4955Crossref PubMed Scopus (35) Google Scholar). For siRNA knockdown of protein expression, the following nucleotide (nt) sequences were cloned into pRNAU6-hygro vector (GenScript, Piscataway, NJ): sequence 1 (5′-acatgagactggtggctat-3′ (NM_019143.1, nt 681-699)) and sequence 2 (5′-aacaaatctcctgcctgggac-3′ (NM_019143.1, nt 4452-4472)) (25Sakai T. Larsen M. Yamada K.M. Nature. 2003; 423: 876-881Crossref PubMed Scopus (406) Google Scholar) for rat Fn1; sequence 3 (5′-atggtagtgttcaatggc-3′ (NM_017171.1, nt 194-211)) (26Cheng J.J. Wung B.S. Chao Y.J. Wang D.L. J. Biol. Chem. 2001; 276: 31368-31375Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and sequence 4 (5′-ccaactctattgctgcttc-3′ (NM_017171.1, nt 1603-1621)) for rat PKCϵ; sequence 5 (5′-aactcgcacatagcgactctg-3′) for the nonspecific control sequence. The siRNA plasmid pKD-PKCδ-v6 was purchased from Upstate. All plasmid constructs were verified by double-stranded sequencing.MTF7L cells grown to 70% confluence were transiently transfected with the above vector constructs or vector alone using Lipofectamine Plus as described by the manufacturer (Invitrogen). Transfection rates assessed by expression of GFP that is either tagged to the cDNA of interest or co-transfected at a ratio of 1:50 with the cDNA of interest were 20-30%. Cells were used in the various assays 48 h after transfection unless otherwise stated. Stable clones were obtained by hygromycin selection (750 μg/ml). In some cases hygromycin-selected clones (cl) were further selected by fluorescence-activated cell sorting (FACS) for optimal expression.Flow Cytometry—FACS was used to quantify Fn expression on MTF7L breast cancer cell surfaces (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Cheng H.C. Abdel-Ghany M. Pauli B.U. J. Biol. Chem. 2003; 278: 24600-24607Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Tumor cells that had been subjected to EoE suspension culture were washed twice in DMEM containing 1% bovine serum albumin (BSA), then incubated with rabbit anti-Fn[pan] antibody diluted 1:100 in PBS containing 1% BSA (PBS-BSA) for 1 h at 4 °C. After washing in PBS-BSA, tumor cells were stained with PE-conjugated donkey anti-rabbit antiserum in PBS-BSA for 1 h at 4 °C and fixed in 2% paraformaldehyde in PBS. In select experiments cells were stained with mouse anti-Fn[EDA] (diluted 1:50) and PE-conjugated goat anti-mouse antiserum. FACS analysis was performed on a Coulter Epics Profile (Coulter Electronics, Hialeah, FL). Nonspecific fluorescence was accounted for by incubating tumor cells with non-immune rabbit serum instead of primary antibody. To quantify the effect of overexpressed or knocked-down proteins on polyFn assembly, we generate bivariate distributions of red fluorescence (y axis: cells stained with anti-Fn antibodies and PE-conjugated secondary antibodies) and green fluorescence (x axis: same cells expressing GFP-tagged protein or co-transfected with GFP and cDNA of interest). The levels of polyFn expression in the cell population that emitted high GFP fluorescence were taken as a reflection of the effect of the transfected cDNA on polyFn assembly. To assess the effect of inhibitors of cell signaling, tumor cells were incubated with inhibitor 30 min before (adherent) and throughout EoE suspension culture, then subjected to polyFn quantification as described above. Controls were tumor cells incubated in equimolar inhibitor solvent concentration.Semiquantitative Reverse Transcription-PCR Analyses—Total RNA was prepared from MTF7L grown as adherent monolayers or in EoE suspension cultures by extraction with Trizol as described by the manufacturer (Invitrogen). For every experimental sample, total RNA was quantified both spectrophotometrically and electrophoretically, and amounts were adjusted so that 1 μg was reverse-transcribed (SuperScript reverse transcriptase; Invitrogen). cDNA was subjected to PCR (93 °C for 30 s; 55 °C, 30 s; 72 °C, 30 s; 35 cycles) using TaqDNA polymerase (Invitrogen) and primer sets derived from rat Fn1 (NM_019143), PKCδ (NM_133307), PKCϵ (NM_017171), PKCη (NM_031085), PKCθ (XM_341553), and PKCζ ((NM_022507). Controls were run in the absence of reverse transcriptase. Glyceraldehyde-3-phosphate dehydrogenase served as reference standard.Cell Fractionation—MTF7L cells and transfectants thereof were incubated in EoE suspension culture in DMEM containing 20% FBS for the indicated periods of time. Cells were washed in PBS, collected by centrifugation, and resuspended in 0.5 ml ice-cold Tris buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 0.1 mm NaVO4, 20 μm leupeptin, 0.1% aprotinin, and 1 mm phenylmethylsulfonyl fluoride). Cells were disrupted by two 15-s cycles of sonication at 4 °C using a microprobe sonicator at maximum power. After removal of unbroken cells and nuclei by centrifugation (500 × g for 5 min at 4 °C), supernatants were centrifuged at 16,300 × g for 15 min at 4 °C. The resulting supernatant was designated the cytosolic fraction. The pellet was solubilized in 0.5 ml of buffer A containing 1% Triton X-100 for 1 h at 4 °C EoE, then centrifuged at 16,300 × g for 15 min at 4 °C. The detergent-solute was designated the membrane fraction. Twenty micrograms of protein from both the cytosolic and membrane fractions were separated by SDS-PAGE (10%), transferred to nitrocellulose membrane at 4 °C, and probed by Western blotting as described (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar).Cell Lysis, Immunoprecipitation, Western Blotting, and Autoradiography—Cells were extracted with lysis Tris buffer containing 1% Triton X-100 for 1 h at 4 °C (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Total cell lysates or cytosolic and membrane fractions were subjected to (i) SDS-PAGE (∼20-50 μg of protein) and Western blotting using anti-Fn[pan], anti-Fn[EDA], or various PKC isoform-specific antibodies, horseradish peroxidase-conjugated donkey anti-rabbit, or goat anti-mouse secondary antibodies and ECL for detection of bound antibody as described (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and (ii) immunoprecipitation with anti-Fn[pan], anti-PKCϵ, or anti-PKCδ antibodies (27Abdel-Ghany M. Cheng H.C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Immunoprecipitates obtained from lysates of unlabeled, [32P]orthophosphate-, or [35S]methionine-labeled cells were separated by SDS-PAGE (6-12% polyacrylamide) and analyzed by autoradiography (radio-labeled samples) or blotted to nitrocellulose membranes and probed with either anti-Fn[pan], anti-Fn[EDA], anti-PKC isoform-specific antibodies, or anti-Ser(P) and anti-Thr(P) antibodies (9Cheng H.C. Abdel-Ghany M. Elble R.C. Pauli B.U. J. Biol. Chem. 1998; 273: 24207-24215Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar).Tumor Cell Proliferation Assay—MTF7L cells and clones thereof were seeded into 96-well microtitration plates (500 cells/well) and incubated in DMEM containing 10% FBS. Cell growth was monitored in daily intervals for up to 4 days. At the end of each incubation period, tumor cells were fixed with 2% paraformaldehyde in PBS and then stained with 0.5% crystal violet in 20% methanol as described (21Cheng H.C. Abdel-Ghany M. Zhang S. Pauli B.U. Clin. Exp. Metastasis. 1999; 17: 609-615Crossref PubMed Scopus (30) Google Scholar). Absorbance was read on a microplate reader (Bio-Teck Instruments) at 562 nm and graphed as a function of time of incubation.Isolation of Blood-borne Cancer Cells from Tumor-bearing Rats—MTF7L cancer cells (1 × 106 cells/50 μl of DMEM) were injected into the 4th (left + right) mammary fat pads of six 6-week-old female Fischer 344 rats. At a tumor diameter of ∼2 cm, rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (65 mg/kg body weight), and blood was collected by cardiac puncture. Pooled, EDTA-treated blood was transferred to precooled 50-ml centrifuge tubes containing 15 ml of OncoQuick tumor enrichment medium below a porous barrier (Greiner Bio-One, Longwood, FL) and centrifuged at 1600 × g for 20 min at 4 °C in a swing-out rotor as described by Rosenberg et al. (28Rosenberg R. Gertler R. Friedrichs J. Fuehrer K. Dahm M. Phelps R. Thorban S. Nekarda H. Siewert J.R. Cytometry. 2002; 49: 150-158Crossref PubMed Scopus (231) Google Scholar). After a second round of centrifugation of the fluid in the upper compartment, cells were washed with PBS-BS

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