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• W1990465778 abstract "Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation, both ligand-dependent (eg, epidermal growth factor treatment) and ligand-independent (eg, overexpression), was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825, in epidermal growth factor-treated C-33 LNCaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together, these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overexpressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion. Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation, both ligand-dependent (eg, epidermal growth factor treatment) and ligand-independent (eg, overexpression), was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825, in epidermal growth factor-treated C-33 LNCaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together, these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overexpressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion. Prostate cancer (PCa) is one of the most common malignancies among men in Western countries and the incidence is progressively rising within low-risk populations including China, Japan, and Taiwan.1Chen CJ You SL Lin LH Hsu WL Yang YW Cancer epidemiology and control in Taiwan: a brief review.Jpn J Clin Oncol. 2002; 32: S66-S81Crossref PubMed Scopus (168) Google Scholar, 2Jemal A Siegel R Ward E Murray T Xu J Thun MJ Cancer statistics, 2007.CA Cancer J Clin. 2007; 57: 43-66Crossref PubMed Scopus (7452) Google Scholar The death of patients with PCa is mainly due to hormone refractory and metastatic disease,3Sporn MB The war on cancer.Lancet. 1996; 347: 1377-1381Abstract Full Text PDF PubMed Scopus (545) Google Scholar and metastatic ability is believed to be due to cancer cells gaining the ability to degrade the extracellular matrix.4Stetler-Stevenson WG Aznavoorian S Liotta LA Tumor cell interactions with the extracellular matrix during invasion and metastasis.Annu Rev Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1521) Google Scholar Overexpression of ErbB-2 has been reported and is believed to participate in oncogenic signaling in metastatic prostate cancer.5Carles J Lloreta J Salido M Font A Suarez M Baena V Nogue M Domenech M Fabregat X Her-2/neu expression in prostate cancer: a dynamic process?.Clin Cancer Res. 2004; 10: 4742-4745Crossref PubMed Scopus (30) Google Scholar, 6Osman I Scher HI Drobnjak M Verbel D Morris M Agus D Ross JS Cordon-Cardo C HER-2/neu (p185neu) protein expression in the natural or treated history of prostate cancer.Clin Cancer Res. 2001; 7: 2643-2647PubMed Google Scholar, 7Shi Y Brands FH Chatterjee S Feng AC Groshen S Schewe J Lieskovsky G Cote RJ Her-2/neu expression in prostate cancer: high level of expression associated with exposure to hormone therapy and androgen independent disease.J Urol. 2001; 166: 1514-1519Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 8Signoretti S Montironi R Manola J Altimari A Tam C Bubley G Balk S Thomas G Kaplan I Hlatky L Hahnfeldt P Kantoff P Loda M Her-2-neu expression and progression toward androgen independence in human prostate cancer.J Natl Cancer Inst. 2000; 92: 1918-1925Crossref PubMed Scopus (331) Google Scholar, 9Zellweger T Ninck C Bloch M Mirlacher M Koivisto PA Helin HJ Mihatsch MJ Gasser TC Bubendorf L Expression patterns of potential therapeutic targets in prostate cancer.Int J Cancer. 2005; 113: 619-628Crossref PubMed Scopus (141) Google Scholar ErbB-2 signaling has been shown to induce several phenotypic changes associated with more aggressive disease such as increased cell motility,10Grothey A Hashizume R Ji H Tubb BE Patrick Jr, CW Yu D Mooney EE McCrea PD C-erbB-2/HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines.Oncogene. 2000; 19: 4864-4875Crossref PubMed Scopus (108) Google Scholar cell proliferation,11Engelsen IB Stefansson IM Beroukhim R Sellers WR Meyerson M Akslen LA Salvesen HB HER-2/neu expression is associated with high tumor cell proliferation and aggressive phenotype in a population based patient series of endometrial carcinomas.Int J Oncol. 2008; 32: 307-316PubMed Google Scholar and/or invasive ability.12Spencer KS Graus-Porta D Leng J Hynes NE Klemke RL ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases.J Cell Biol. 2000; 148: 385-397Crossref PubMed Scopus (157) Google Scholar Although no direct ligand for ErbB-2 has yet been identified, ErbB-2 activation is known to occur in both a ligand-independent and ligand-dependent manner.13Dawson JP Bu Z Lemmon MA Ligand-induced structural transitions in ErbB receptor extracellular domains.Structure. 2007; 15: 942-954Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 14Garrett TP McKern NM Lou M Elleman TC Adams TE Lovrecz GO Kofler M Jorissen RN Nice EC Burgess AW Ward CW The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors.Mol Cell. 2003; 11: 495-505Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 15Lemmon MA Ligand-induced ErbB receptor dimerization.Exp Cell Res. 2009; 315: 638-648Crossref PubMed Scopus (161) Google Scholar, 16Olayioye MA Neve RM Lane HA Hynes NE The ErbB signaling network: receptor heterodimerization in development and cancer.EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar Ligand-dependent ErbB-2 activation involves heterodimerization of the receptor with another ligand-activated ErbB receptor (epidermal growth factor [EGF] receptor/ErbB-1, ErbB-3, and ErbB-4).17Riese 2nd, DJ Stern DF Specificity within the EGF family/ErbB receptor family signaling network.Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (697) Google Scholar, 18Yarden Y Sliwkowski MX Untangling the ErbB signalling network.Nat Rev Mol Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5609) Google Scholar For example, in prostate cancer cells, the ErbB ligand EGF induces the formation of both ErbB-1/ErbB-1 homodimers and ErbB-1/ErbB-2 heterodimers.19El Sheikh SS Domin J Abel P Stamp G Lalani el N Phosphorylation of both EGFR and ErbB2 is a reliable predictor of prostate cancer cell proliferation in response to EGF.Neoplasia. 2004; 6: 846-853Abstract Full Text PDF PubMed Scopus (77) Google Scholar Ligand-independent ErbB-2 activation is largely thought to be due to ErbB-2 overexpression, resulting in spontaneous homodimerization and autoactivation.20Citri A Yarden Y EGF-ERBB signalling: towards the systems level.Nat Rev Mol Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1594) Google Scholar, 21Hynes NE Lane HA ERBB receptors and cancer: the complexity of targeted inhibitors.Nat Rev Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2679) Google Scholar Clinical data show that ErbB-2 protein levels are elevated in a subset of patients with clinically significant prostate cancer, especially in the recurrent, hormone-refractory phase of the disease.6Osman I Scher HI Drobnjak M Verbel D Morris M Agus D Ross JS Cordon-Cardo C HER-2/neu (p185neu) protein expression in the natural or treated history of prostate cancer.Clin Cancer Res. 2001; 7: 2643-2647PubMed Google Scholar, 8Signoretti S Montironi R Manola J Altimari A Tam C Bubley G Balk S Thomas G Kaplan I Hlatky L Hahnfeldt P Kantoff P Loda M Her-2-neu expression and progression toward androgen independence in human prostate cancer.J Natl Cancer Inst. 2000; 92: 1918-1925Crossref PubMed Scopus (331) Google Scholar, 22Montironi R Mazzucchelli R Barbisan F Stramazzotti D Santinelli A Scarpelli M Lopez Beltran A HER2 expression and gene amplification in pT2a Gleason score 6 prostate cancer incidentally detected in cystoprostatectomies: comparison with clinically detected androgen-dependent and androgen-independent cancer.Hum Pathol. 2006; 37: 1137-1144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar ErbB-2 overexpression promotes the survival and growth of prostate cancer cells through the MAPK and/or phosphatidylinositol 3-kinase (PI3K)/AKT pathway in the absence of androgens,8Signoretti S Montironi R Manola J Altimari A Tam C Bubley G Balk S Thomas G Kaplan I Hlatky L Hahnfeldt P Kantoff P Loda M Her-2-neu expression and progression toward androgen independence in human prostate cancer.J Natl Cancer Inst. 2000; 92: 1918-1925Crossref PubMed Scopus (331) Google Scholar, 23Wen Y Hu MC Makino K Spohn B Bartholomeusz G Yan DH Hung MC HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway.Cancer Res. 2000; 60: 6841-6845PubMed Google Scholar and abnormal ErbB-2 activation due to overexpression may serve as one of the key factors leading to prostate cancer recurrence during hormone ablation therapy, and/or to promote prostate tumors with a highly metastatic potential.8Signoretti S Montironi R Manola J Altimari A Tam C Bubley G Balk S Thomas G Kaplan I Hlatky L Hahnfeldt P Kantoff P Loda M Her-2-neu expression and progression toward androgen independence in human prostate cancer.J Natl Cancer Inst. 2000; 92: 1918-1925Crossref PubMed Scopus (331) Google Scholar, 24Scher HI HER2 in prostate cancer: a viable target or innocent bystander?.J Natl Cancer Inst. 2000; 92: 1866-1868Crossref PubMed Scopus (35) Google Scholar Thus, ErbB-2 overexpression in patients with prostate cancer has been associated with poor prognosis.25Hernes E Fossa SD Berner A Otnes B Nesland JM Expression of the epidermal growth factor receptor family in prostate carcinoma before and during androgen-independence.Br J Cancer. 2004; 90: 449-454Crossref PubMed Scopus (146) Google Scholar The exact nature of the contribution made by elevated ErbB-2 protein levels to prostate cancer cell invasion during the cancer progression is, however, still not well-characterized. Cell surface proteolysis by secreted or plasma membrane-anchored proteases has been strongly implicated in cancer cell invasion and metastasis due to their roles in the degradation of the Extracellular matrix, cell growth, adhesion, and migration.26Werb Z ECM and cell surface proteolysis: regulating cellular ecology.Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar Recently, several lines of evidence have shown that deregulation of type II transmembrane serine proteases may play a role in many diseases and, in particular, may enhance tumor growth, invasion, and metastasis.27Netzel-Arnett S Hooper JD Szabo R Madison EL Quigley JP Bugge TH Antalis TM Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer.Cancer Metastasis Rev. 2003; 22: 237-258Crossref PubMed Scopus (256) Google Scholar, 28Dano K Romer J Nielsen BS Bjorn S Pyke C Rygaard J Lund LR Cancer invasion and tissue remodeling: cooperation of protease systems and cell types.Apmis. 1999; 107: 120-127Crossref PubMed Scopus (289) Google Scholar, 29Johnsen M Lund LR Romer J Almholt K Dano K Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.Curr Opin Cell Biol. 1998; 10: 667-671Crossref PubMed Scopus (328) Google Scholar, 30Kohn EC Liotta LA Molecular insights into cancer invasion: strategies for prevention and intervention.Cancer Res. 1995; 55: 1856-1862PubMed Google Scholar, 31Chen WT Proteases associated with invadopodia, and their role in degradation of extracellular matrix.Enzyme Protein. 1996; 49: 59-71Crossref PubMed Scopus (103) Google Scholar One of the best characterized type II transmembrane serine proteases is the enzyme matriptase.32Lin CY Wang JK Torri J Dou L Sang QA Dickson RB Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells: monoclonal antibody production, isolation, and localization.J Biol Chem. 1997; 272: 9147-9152Crossref PubMed Scopus (120) Google Scholar, 33Cao J Cai X Zheng L Geng L Shi Z Pao CC Zheng S Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening.J Cancer Res Clin Oncol. 1997; 123: 447-451Crossref PubMed Scopus (41) Google Scholar, 34Kim MG Chen C Lyu MS Cho EG Park D Kozak C Schwartz RH Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains.Immunogenetics. 1999; 49: 420-428Crossref PubMed Scopus (124) Google Scholar, 35Takeuchi T Shuman MA Craik CS Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.Proc Natl Acad Sci USA. 1999; 96: 11054-11061Crossref PubMed Scopus (231) Google Scholar, 36Tanimoto H Underwood LJ Wang Y Shigemasa K Parmley TH O'Brien TJ Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention.Tumour Biol. 2001; 22: 104-114Crossref PubMed Scopus (75) Google Scholar In human cancer, matriptase is expressed in a variety of epithelial-derived tumors including prostate, breast, colon, stomach, and ovarian carcinomas.35Takeuchi T Shuman MA Craik CS Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.Proc Natl Acad Sci USA. 1999; 96: 11054-11061Crossref PubMed Scopus (231) Google Scholar, 36Tanimoto H Underwood LJ Wang Y Shigemasa K Parmley TH O'Brien TJ Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention.Tumour Biol. 2001; 22: 104-114Crossref PubMed Scopus (75) Google Scholar, 37Oberst M Anders J Xie B Singh B Ossandon M Johnson M Dickson RB Lin CY Matriptase and HAI-1 are expressed by normal and malignant epithelial cells in vitro and in vivo.Am J Pathol. 2001; 158: 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 38Santin AD Cane S Bellone S Bignotti E Palmieri M De Las Casas LE Anfossi S Roman JJ O'Brien T Pecorelli S The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma.Cancer. 2003; 98: 1898-1904Crossref PubMed Scopus (52) Google Scholar In the prostate, matriptase expression is increased in primary prostate cancers and in metastatic lesions.39Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1429) Google Scholar Immunohistochemical analysis has shown that increased matriptase protein level is correlated with tumor grade and poor prognosis.40Saleem M Adhami VM Zhong W Longley BJ Lin CY Dickson RB Reagan-Shaw S Jarrard DF Mukhtar H A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1.Cancer Epidemiol Biomarkers Prev. 2006; 15: 217-227Crossref PubMed Scopus (126) Google Scholar Moreover, a role for matriptase in carcinogenesis is further supported by data from animal models. Overexpression of matriptase in the skin of transgenic mice, driven by a keratin-5 promoter, induces ras-independent multistage carcinogenesis and promotes ras-mediated, carcinogen-induced tumor formation.41List K Szabo R Molinolo A Sriuranpong V Redeye V Murdock T Burke B Nielsen BS Gutkind JS Bugge TH Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.Genes Dev. 2005; 19: 1934-1950Crossref PubMed Scopus (211) Google Scholar Recently, matriptase has been suggested to be involved in the activation process of the pro-oncogenic and prometastatic factors pro-uPA and pro-HGF/SF.42Forbs D Thiel S Stella MC Sturzebecher A Schweinitz A Steinmetzer T Sturzebecher J Uhland K In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma.Int J Oncol. 2005; 27: 1061-1070PubMed Google Scholar, 43Kang JY Dolled-Filhart M Ocal IT Singh B Lin CY Dickson RB Rimm DL Camp RL Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer.Cancer Res. 2003; 63: 1101-1105PubMed Google Scholar, 44Suzuki M Kobayashi H Kanayama N Saga Y Suzuki M Lin CY Dickson RB Terao T Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase.J Biol Chem. 2004; 279: 14899-14908Crossref PubMed Scopus (68) Google Scholar, 45Lee SL Dickson RB Lin CY Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease.J Biol Chem. 2000; 275: 36720-36725Crossref PubMed Scopus (354) Google Scholar Despite these varied observations, little is known about the role of oncogenic signaling in matriptase activation or how this might be involved in enhanced cancer malignancy during the progression of prostate cancer. In this study, we explore the possible role that ErbB-2 signaling may play in the deregulation of cell-surface proteolysis through modulation of matriptase zymogen activation, and how the ErbB-2-matriptase axis may promote an invasive phenotype in prostate cancer cells. Lipofectamine 2000 transfection reagent and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium, and RPMI 1640 culture media were obtained from Hyclone (Logan, UT). Protein assay kits were from Bio-Rad (Hercules, CA). Protein molecular weight standard markers and an anti-Myc epitope antibody (Ab) were obtained from Bioman Scientific Company, Ltd (Taipei, Taiwan). Polyclonal anti-ErbB-2 Ab (C-18) for immunoblotting and horseradish peroxidase-conjugated anti-rabbit IgG Ab were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine (pY100), anti-phospho-p44/p42 ERK1/2 (Thr202/Tyr204), rabbit polyclonal anti-phospho-ErbB-2 (Y877), rabbit polyclonal anti-phospho-ErbB-2 (Y1221/Y1222), rabbit polyclonal anti-phospho-ErbB-2 (Y1248), anti-phospho Akt, and anti-Akt Abs were obtained from Cell Signaling Technology (Beverly, MA), and a polyclonal anti-Erk1/2 Ab was purchased from Millipore (Millipore, CA). The mouse monoclonal anti-phosphotyrosine (4G10) Ab was purchased from Upstate Biotechnology (Lake Placid, NY). The Enhanced Chemiluminescence (ECL) detection system was purchased from Pierce (Rockford, IL). AG825, LY294002, and PD98059 were from Calbiochem (San Diego, CA). EGF, heregulin-β1, and all other chemicals and reagents were purchased from Sigma (St. Louis, MO), unless otherwise noted. The human prostate carcinoma cell lines DU145, C-33, and C-81 LNCaP cells were gifts from Dr. Ming-Fong Lin at the Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center (Omaha, NE). C-33 cells, C-81 cells, and DU 145 cells were maintained in a regular medium consisting of RPMI 1640 supplemented with 5% FBS, 1% glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. HEK 293T cells were used for lentiviral production. HEK 293T cells and PC-3 cells were gifts from Dr. Pei-Wen Hsiao, Institute of Agricultural Biotechnology Research Center, Academia Sinica, Taiwan, and cultured in Dulbecco's modified Eagle's medium with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded in regular culture medium at a density of 4 × 105 cells for C-81 LNCaP cells and 6 × 105 cells for C-33 LNCaP cells per 60-mm dish. One day after plating, transient transfection with wild-type ErbB-2, constitutively active MEK, or Akt plasmids was performed by Lipofectamine 2000 reagent in Optimum-Minimum Essential Medium (Invitrogen, Carlsbad, CA) medium, according to the manufacturer's instruction. The transfected cells were harvested 48 hours after transfection. The ErbB-2 and a constitutively active MEK expression constructs were gifts from Dr. Ming-Fong Lin.46Meng TC Lee MS Lin MF Interaction between protein tyrosine phosphatase and protein tyrosine kinase is involved in androgen-promoted growth of human prostate cancer cells.Oncogene. 2000; 19: 2664-2677Crossref PubMed Scopus (64) Google Scholar, 47Lee MS Igawa T Yuan TC Zhang XQ Lin FF Lin MF ErbB-2 signaling is involved in regulating PSA secretion in androgen-independent human prostate cancer LNCaP C-81 cells.Oncogene. 2003; 22: 781-796Crossref PubMed Scopus (54) Google Scholar The Myr-Akt plasmid was a gift from Dr. Ruey-Hwa Chen, Institute of Biological Chemistry, Academia Sinica, Taiwan. LNCaP cells (1 × 107 cells) were washed with PBS and a lysate was prepared by adding 1 ml of immunoprecipitation assay buffer (50 mmol/L Tris–HCl, pH 7.8, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.1% deoxycholate, and 10 μg/ml each of leupeptin, aprotinin, and phenylmethylsulfonyl fluoride) to the cells. The lysate was then centrifuged by using a microcentrifuge at 10,000 × g for 20 minutes. The supernatant was added to an anti-ErbB-2 Ab at 4°C for 1 hour. Protein-A/G-agarose beads (30 μl; Calbiochem) were added to the lysate, and the mixture was incubated with shaking for 1 hour at 4°C. The beads were finally collected by centrifugation and washed three times with immunoprecipitation assay buffer. Proteins binding to the beads were eluted by adding 20 μl of 2× electrophoresis sample buffer and analyzed by immunoblotting with an anti-phosphotyrosine Ab (4G10). After analysis the membranes were stripped and reblotted with an anti-ErbB-2 Ab to detect total protein levels. Two days after transient transfection with the ErbB-2 plasmid or parent vector alone, the cells were treated with 400 μg/ml Geneticin/G418 to select for stable integration of the plasmids. Stable pools of ErbB-2 or control transfected cells were maintained in a regular medium supplemented with 400 μg/ml of Geneticin. Small hairpin RNAs (shRNA) in a lentiviral infection system were obtained from the National RNAi Core Facility of Academia Sinica, Nankang, Taiwan. shRNAs for the knockdown of ErbB-2 (shErbB-2, clone ID: TRCN 0000039879; shErbB-2u, clone ID: TRCN 0000303527), Akt1 (shAkt1-1, clone ID: TRCN 0000039795; Akt1-2, clone ID: TRCN 0000010174), Akt2 (shAkt2-1, clone ID: TRCN 0000265834; Akt2-2, clone ID: TRCN 0000255915), matriptase (shMTX, clone ID: TRCN 0000038053), or a negative control shRNA for knockdown of luciferase (shLuc) were all in the pLKO.1-puro vector and were packaged into lentiviral particles by using HEK 293T cells by co-transfection with pCMVΔR8.91 and pMD.G, using Lipofectamine 2000, according to the recommended protocol. Conditioned medium from the transfected cells containing lentiviral particles was collected 24 and 48 hours after the addition of fresh medium. For lentiviral infection, cells were seeded at up to 70% confluence and cultured for 24 hours. Lentiviral infection was performed by adding 10% (v/v) of lentivirus-containing medium to the cell culture. Twenty-four hours after infection, transduced cells were selected by exposure to 1 μg/ml puromycin for 3 days. After washing twice with cold PBS (50 mmol/L potassium phosphate and 150 mmol/L NaCl, pH 7.2), the cells were lyzed in a standard lysis buffer (1% Triton X-100 in PBS) or a lysis buffer containing phosphatase and protease inhibitors (20 mmol/L HEPES [pH = 7.0], 0.5% NP-40, 2 mmol/L sodium vanadate, and 1× proteinase inhibitor cocktail; Roche Diagnostics GmbH, Mannheim, Germany). The lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatants were collected. Protein concentrations in the cell lysates were determined by Protein Assay solution (BioRad) with reference to a bovine serum albumin standard curve. Equal amounts of total protein were then mixed with Laemmli sample buffer for electrophoresis. Generally, 5% β-mercaptoethanol was added to the samples, which were then boiled for 10 minutes before loading on the gels; however, when blotting for matriptase or HAI-1, this was not done, because boiling disrupts matriptase-HAI-1 complexes, and reducing agents destroy the epitopes recognized by the monoclonal antibodies (mAbs).48Oberst MD Williams CA Dickson RB Johnson MD Lin CY The activation of matriptase requires its noncatalytic domains, serine protease domain, and its cognate inhibitor.J Biol Chem. 2003; 278: 26773-26779Crossref PubMed Scopus (156) Google Scholar Fifty micrograms of proteins per well were loaded and separated by SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes with a transfer buffer consisting of 25 mmol/L Tris-base, 192 mmol/L glycine, and 20% methanol. After protein transfer and between antibody incubations, the membranes were washed with Tris-Buffered Saline Tween-20 (TBST) wash buffer (20 mmol/L Tris-HCl [pH 7.5], 0.87% [w/v] NaCl, and 0.1% Tween 20). The membranes were blocked with 5% skim milk in TBST, and the specific proteins were recognized by primary antibodies and subsequently by secondary antibodies conjugated with Horseradish peroxidase (Jackson, PA) in 5% skim milk. The target proteins were revealed by enhanced chemiluminescent reagents (Pierce) and detected by X-ray films or a luminescent image analyzer with a CCD camera (LAS-4000; Fujifilm, Tokyo, Japan). Cells for wound healing assays were cultured for 5 to 6 days in 60-mm Petri dishes until fully confluent. Wound gaps were made by scraping cell monolayers with a 10-μl pipette tip, after which the cells were washed with PBS twice and fed with fresh culture medium. Images of wound gaps were taken by a light microscope (TS100; Nikon, Taipei, Taiwan) at 100× and the cells were incubated in a humidified, 37°C, 5% CO2 incubator until subsequent images were taken. Widths of the wound and migratory distances were measured and calculated by using NIS-Elements D software (Nikon). Each observation was carried out in triplicate. Transwell assays were performed according to previously described procedures with slight modification.49Albini A Iwamoto Y Kleinman HK Martin GR Aaronson SA Kozlowski JM McEwan RN A rapid in vitro assay for quantitating the invasive potential of tumor cells.Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar In brief, cell culture inserts (Millipore) were placed in the wells of 24-well plates (Corning Incorporated, Corning, NY). For an invasion assay, 10 μg of Matrigel (BD Biosciences, San Jose, CA) diluted in 100 μl distilled water was added to the filer of each insert, which was then air-dried overnight. The dried Matrigel was reconstituted with 100 μl serum-free RPMI 1640 medium for 1 hour before usage. No Matrigel was coated on filters for migration assay. Cells were serum-starved for 24 hours and then seeded in the upper chambers of transwell with 200 μl serum-free RPMI 1640 medium. The lower chambers were filled with 600 μl RPMI 1640 medium containing 10% FBS as a chemoattractant. After incubation for 48 hours, cells that had penetrated to the external surface of the filters were fixed in methanol for 10 minutes and stained with 1% crystal violet for 10 minutes. After washing with distilled water, the nonpenetrating cells on the in" @default.
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• W1990465778 date "2010-12-01" @default.
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• W1990465778 title "Matriptase Is Involved in ErbB-2-Induced Prostate Cancer Cell Invasion" @default.
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• W1990465778 doi "https://doi.org/10.2353/ajpath.2010.100228" @default.
• W1990465778 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2993294" @default.
• W1990465778 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20971737" @default.
• W1990465778 hasPublicationYear "2010" @default.
• W1990465778 type Work @default.

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