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• W2006733013 abstract "The effect of interferon-γ (IFNγ) treatment on cell surface protein expression was studied in the human prostate cancer cell line 1542CP3TX. IFNγ increased both the number and abundance of proteins in membrane fractions. In contrast, the expression of annexin 2 and its binding partner p11 decreased by 4-fold after 24 h of exposure, with the remaining anx2t complexes localized to lipid rafts. Within the same time scale, IFNγ reduced the abundance of the peripherally attached, anx2t-associated proteases procathepsin B and plasminogen. The invasive capacity of the cancer cells was reduced by treatment with IFNγ or antibody to annexin 2 in 1542CP3TX cells, but not in LNCaP, an annexin 2-negative prostate cancer cell line. Expression of annexin 2 in LNCaP cells increased their invasiveness. IFNγ induced calpain expression and activation and increased the phosphorylation and degradation of the calpain substrate ABCA1 in 1542CP3TX cancer cells. Surface expression of annexin 2 was reduced in cells treated with glyburide, an ABCA1 inhibitor, whereas inhibition of calpain abrogated IFNγ-induced annexin 2 down-regulation and suppression of Matrigel invasion. The findings suggest annexin 2 externalization is coupled to lipid efflux in prostate epithelium and that IFNγ induces down-regulation of the protease-binding anx2t scaffold at the cell surface and consequently acts to suppress invasiveness through calpain-mediated degradation of the lipid transporter ABCA1. The effect of interferon-γ (IFNγ) treatment on cell surface protein expression was studied in the human prostate cancer cell line 1542CP3TX. IFNγ increased both the number and abundance of proteins in membrane fractions. In contrast, the expression of annexin 2 and its binding partner p11 decreased by 4-fold after 24 h of exposure, with the remaining anx2t complexes localized to lipid rafts. Within the same time scale, IFNγ reduced the abundance of the peripherally attached, anx2t-associated proteases procathepsin B and plasminogen. The invasive capacity of the cancer cells was reduced by treatment with IFNγ or antibody to annexin 2 in 1542CP3TX cells, but not in LNCaP, an annexin 2-negative prostate cancer cell line. Expression of annexin 2 in LNCaP cells increased their invasiveness. IFNγ induced calpain expression and activation and increased the phosphorylation and degradation of the calpain substrate ABCA1 in 1542CP3TX cancer cells. Surface expression of annexin 2 was reduced in cells treated with glyburide, an ABCA1 inhibitor, whereas inhibition of calpain abrogated IFNγ-induced annexin 2 down-regulation and suppression of Matrigel invasion. The findings suggest annexin 2 externalization is coupled to lipid efflux in prostate epithelium and that IFNγ induces down-regulation of the protease-binding anx2t scaffold at the cell surface and consequently acts to suppress invasiveness through calpain-mediated degradation of the lipid transporter ABCA1. Prostate cancer is the second most common cause of cancer death in men and the most frequently diagnosed malignancy in men. Whereas the cancer is contained within the prostate, the disease is curable with surgery or radiotherapy, but once it has spread there are no curative treatments. Thus a major challenge is to identify new methods to delay or prevent the progression of the disease. Cytokines are among the most potent extracellular regulators of protein expression at the cell surface. In addition to regulating major histocompatibility complex processing and presentation pathways, interferon-γ (IFNγ) 2The abbreviations used are: IFNγ, interferon-γ; OGP, octyl-β-d-glucopyranoside; PBS, phosphate-buffered saline; GFP, green fluorescent protein; LMW, low molecular weight. up-regulates the surface densities of many molecules (1Amrani Y. Moore P.E. Hoffman R. Shore S.A. Panettieri Jr., R.A. Am. J. Respir. Crit. Care Med. 2001; 164: 2098-2101Crossref PubMed Scopus (72) Google Scholar, 2Anderson F. Game B.A. Atchley D. Xu M. Lopes-Virella M.F. Huang Y. Clin. Immunol. 2002; 102: 200-207Crossref PubMed Scopus (7) Google Scholar, 3Flieger D. Hoff A.S. Sauerbruch T. Schmidt-Wolf I.G. Clin. Exp. Immunol. 2001; 123: 9-14Crossref PubMed Scopus (29) Google Scholar, 4Ibrahim E.C. Guerra N. Lacombe M.J. Angevin E. Chouaib S. Carosella E.D. Caignard A. Paul P. Cancer Res. 2001; 61: 6838-6845PubMed Google Scholar, 5Kaul M. Loos M. FEBS Lett. 2001; 500: 91-98Crossref PubMed Scopus (33) Google Scholar, 6Miller A. Kraiem Z. Sobel E. Lider O. Lahat N. Thyroid. 2000; 10: 945-950Crossref PubMed Scopus (5) Google Scholar, 7Mita Y. Dobashi K. Shimizu Y. Nakazawa T. Mori M. Immunol. Lett. 2001; 78: 97-101Crossref PubMed Scopus (54) Google Scholar, 8Wang X.Q. Evans G.F. Alfaro M.L. Zuckerman S.H. Biochem. Biophys. Res. Commun. 2002; 290: 891-897Crossref PubMed Scopus (14) Google Scholar) and down-regulates the expression of other surface proteins, including CCL20 receptor CCR6 (9Dieu-Nosjean M.C. Massacrier C. Vanbervliet B. Fridman W.H. Caux C. J. Immunol. 2001; 167: 5594-5602Crossref PubMed Scopus (39) Google Scholar), macrophage CD9 (8Wang X.Q. Evans G.F. Alfaro M.L. Zuckerman S.H. Biochem. Biophys. Res. Commun. 2002; 290: 891-897Crossref PubMed Scopus (14) Google Scholar), transferrin receptor (10Ryu S.Y. Jeong K.S. Kang B.N. Park S.J. Yoon W.K. Kim S.H. Kim T.H. Anticancer Res. 2000; 20: 3331-3338PubMed Google Scholar), and interleukin-4 receptor (11So E.Y. Park H.H. Lee C.E. J. Immunol. 2000; 165: 5472-5479Crossref PubMed Scopus (77) Google Scholar). In vitro studies of prostate cancer cell lines have demonstrated that interferons can reduce the growth rate (12Yan D.H. Wen Y. Spohn B. Choubey D. Gutterman J.U. Hung M.C. Oncogene. 1999; 18: 807-811Crossref PubMed Scopus (37) Google Scholar), HER-2 expression (13Kominsky S.L. Hobeika A.C. Lake F.A. Torres B.A. Johnson H.M. Cancer Res. 2000; 60: 3904-3908PubMed Google Scholar), basic fibroblast growth factor expression (14Singh R.K. Gutman M. Bucana C.D. Sanchez R. Llansa N. Fidler I.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4562-4566Crossref PubMed Scopus (420) Google Scholar), and up-regulate p21 WAF1 (15Hobeika A.C. Etienne W. Cruz P.E. Subramaniam P.S. Johnson H.M. Int. J. Cancer. 1998; 77: 138-145Crossref PubMed Scopus (40) Google Scholar, 16Hobeika A.C. Subramaniam P.S. Johnson H.M. Oncogene. 1997; 14: 1165-1170Crossref PubMed Scopus (90) Google Scholar). IFNγ reduced tumor uptake, growth, and metastasis in an experimental mouse model of prostate cancer (17Street S.E. Cretney E. Smyth M.J. Blood. 2001; 97: 192-197Crossref PubMed Scopus (431) Google Scholar), and systemic administration of interferons, in combination with other agents in phase I and II trials, has produced biochemical responses (lowering of serum levels of prostate-specific antigen) in patients with hormonerelapsed prostate cancer (18DiPaola R.S. Rafi M.M. Vyas V. Toppmeyer D. Rubin E. Patel J. Goodin S. Medina M. Medina P. Zamek R. Zhang C. White E. Gupta E. Hait W.N. J. Clin. Oncol. 1999; 17: 2213-2218Crossref PubMed Google Scholar, 19Kramer G. Steiner G.E. Sokol P. Handisurya A. Klingler H.C. Maier U. Foldy M. Marberger M. J. Interferon Cytokine Res. 2001; 21: 475-484Crossref PubMed Scopus (33) Google Scholar, 20Shinohara N. Demura T. Matsumura K. Toyoda K. Kashiwagi A. Nagamori S. Ohmuro H. Ohzono S. Koyanagi T. Prostate. 1998; 35: 56-62Crossref PubMed Scopus (15) Google Scholar, 21Slovin S.F. Scher H.I. Divgi C.R. Reuter V. Sgouros G. Moore M. Weingard K. Pettengall R. Imbriaco M. El-Shirbiny A. Finn R. Bronstein J. Brett C. Milenic D. Dnistrian A. Shapiro L. Schlom J. Larson S.M. Clin. Cancer Res. 1998; 4: 643-651PubMed Google Scholar). Annexin 2 is a member of a family of peripheral membrane-binding proteins characterized by their ability to bind to acidic phospholipids in a calcium-dependent manner. This property is shared with two other protein families, namely the pentraxins and vitamin-K-dependent proteins (22Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1634) Google Scholar). Annexin 2 is unique within the annexin family, as it exists both as a monomer and in a heterotetrameric complex within cells (22Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1634) Google Scholar). The tetrameric complex is formed by two copies of the 36-kDa annexin 2 molecule bound to a dimer of the p11 protein, a member of the S-100 family of calcium-binding proteins, also referred to as the annexin 2 light chain (23Glenney Jr., J.R. Kindy M.S. Zokas L. J. Cell Biol. 1989; 108: 569-578Crossref PubMed Scopus (88) Google Scholar). Annexin 2 is found on the surface of many cell types, including neurons, leukocytes, monocytes, macrophages, and endothelial cells (24Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (208) Google Scholar, 25Falcone D.J. Borth W. Khan K.M. Hajjar K.A. Blood. 2001; 97: 777-784Crossref PubMed Scopus (90) Google Scholar, 26Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 27Jacovina A.T. Zhong F. Khazanova E. Lev E. Deora A.B. Hajjar K.A. J. Biol. Chem. 2001; 276: 49350-49358Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Ma K. Simantov R. Zhang J.C. Silverstein R. Hajjar K.A. Mc-Crae K.R. J. Biol. Chem. 2000; 275: 15541-15548Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 29Menell J.S. Cesarman G.M. Jacovina A.T. McLaughlin M.A. Lev E.A. Hajjar K.A. N. Engl. J. Med. 1999; 340: 994-1004Crossref PubMed Scopus (321) Google Scholar). Surface-bound annexin 2 interacts with extracellular matrix proteins such as collagen 1 (30Mai J. Waisman D.M. Sloane B.F. Biochim. Biophys. Acta. 2000; 1477: 215-230Crossref PubMed Scopus (169) Google Scholar) and tenascin-C (24Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (208) Google Scholar), and mediates high affinity binding of β2-glycoprotein I to endothelial cells (28Ma K. Simantov R. Zhang J.C. Silverstein R. Hajjar K.A. Mc-Crae K.R. J. Biol. Chem. 2000; 275: 15541-15548Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The tetrameric annexin 2-p11 complex also functions as a receptor for tissue-type plasminogen activator and plasminogen, and their simultaneous binding to annexin 2/p11 at the endothelial cell surface results in a 60-fold increase in the catalytic efficiency of plasmin generation (26Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 31Brownstein C. Falcone D.J. Jacovina A. Hajjar K.A. Ann. N. Y. Acad. Sci. 2001; 947: 143-155Crossref PubMed Scopus (31) Google Scholar, 32Fitzpatrick S.L. Kassam G.K. Choi K.S. Kang H.M. Fogg D.K. Waisman D.M. Biochemistry. 2000; 39: 1021-1028Crossref PubMed Scopus (43) Google Scholar, 33Mijung Kwon T.J.M. Zhang Y. Morton Waisman D. Front. Biosci. 2005; 10: 300-325Crossref PubMed Scopus (150) Google Scholar). Increased production of the fibrinolytic serine protease plasmin by annexin 2-overexpressing leukocytes is associated with hemorrhagic complications in patients with acute promyelocytic leukemia (29Menell J.S. Cesarman G.M. Jacovina A.T. McLaughlin M.A. Lev E.A. Hajjar K.A. N. Engl. J. Med. 1999; 340: 994-1004Crossref PubMed Scopus (321) Google Scholar, 34Rand J.H. Biochim. Biophys. Acta. 2000; 1498: 169-173Crossref PubMed Scopus (69) Google Scholar). Annexin 2-mediated plasmin generation further facilitates matrix degradation and invasion by macrophages (25Falcone D.J. Borth W. Khan K.M. Hajjar K.A. Blood. 2001; 97: 777-784Crossref PubMed Scopus (90) Google Scholar) and neurite development in differentiating PC-12 cells (27Jacovina A.T. Zhong F. Khazanova E. Lev E. Deora A.B. Hajjar K.A. J. Biol. Chem. 2001; 276: 49350-49358Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Up-regulation of annexin 2 on the surface of tumor cells has been reported in colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, malignant melanoma, and glioblastoma multiforme (35Emoto K. Yamada Y. Sawada H. Fujimoto H. Ueno M. Takayama T. Kamada K. Naito A. Hirao S. Nakajima Y. Cancer. 2001; 92: 1419-1426Crossref PubMed Scopus (149) Google Scholar, 36Heinzel S. Rea D. Offringa R. Pawelec G. Cancer Immunol. Immunother. 2001; 49: 671-678Crossref PubMed Scopus (13) Google Scholar, 37Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. Chem. 2000; 275: 12806-12812Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and can be associated with poor prognosis (35Emoto K. Yamada Y. Sawada H. Fujimoto H. Ueno M. Takayama T. Kamada K. Naito A. Hirao S. Nakajima Y. Cancer. 2001; 92: 1419-1426Crossref PubMed Scopus (149) Google Scholar, 37Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. Chem. 2000; 275: 12806-12812Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Activated leukocyte cell adhesion molecule and annexin 2 are implicated in the metastatic progression of tumor cells after chemotherapy with adriamycin (38Choi S. Kobayashi M. Wang J. Habelhah H. Okada F. Hamada J. Moriuchi T. Totsuka Y. Hosokawa M. Clin. Exp. Metastasis. 2000; 18: 45-50Crossref PubMed Scopus (40) Google Scholar), and autoantibodies to annexin 2 are frequently observed in patients with lung cancer (39Brichory F.M. Misek D.E. Yim A.M. Krause M.C. Giordano T.J. Beer D.G. Hanash S.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9824-9829Crossref PubMed Scopus (273) Google Scholar). Annexin 2 serves as a binding platform for procathepsin B on the surface of tumor cells (37Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. Chem. 2000; 275: 12806-12812Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and expression of annexin 2 facilitates tissue plasminogen activator-dependent, plasmin-mediated invasion in breast and pancreatic cancers (40Sharma M.R. Koltowski L. Ownbey R.T. Tuszynski G.P. Sharma M.C. Exp. Mol. Pathol. 2006; 81: 146-156Crossref PubMed Scopus (189) Google Scholar, 41Díaz V.M. Hurtado M. Thomson T.M. Reventós J. Paciucci R. Gut. 2004; 53: 993-1000Crossref PubMed Scopus (107) Google Scholar). We have used a proteomics based strategy to identify new cell surface markers in prostate cancer and have identified several deregulated membrane proteins by comparing their expression and regulation by cytokines in normal and cancer cells (42Hastie C. Saxton M. Akpan A. Cramer R. Masters J.R. Naaby-Hansen S. Oncogene. 2005; 24: 5905-5913Crossref PubMed Scopus (44) Google Scholar, 43Nagano K. Masters J.R. Akpan A. Yang A. Corless S. Wood C. Hastie C. Zvelebil M. Cramer R. Naaby-Hansen S. Oncogene. 2003; 23: 1693-1704Crossref Scopus (48) Google Scholar). Here, long term treatment with IFNγ is shown to reduce the surface expression of annexin 2 and its binding partner p11 in 1542CP3TX prostate cancer cells. In consequence, the abundance of surface associated pre-proteases such as procathepsin B and plasminogen is reduced, as is invasiveness. This study demonstrates a novel anticancer action of IFNγ. Cells—The prostate cancer cell line 1542CP3TX was grown in keratinocyte serum-free medium supplemented with 5% fetal calf serum, as described by the originators (44Bright R.K. Vocke C.D. Emmert-Buck M.R. Duray P.H. Solomon D. Fetsch P. Rhim J.S. Linehan W.M. Topalian S.L. Cancer Res. 1997; 57: 995-1002PubMed Google Scholar). LNCaP cells were grown in RPMI 1640 medium containing 10% fetal calf serum. Surface Labeling, Enrichment, and Separation of Membrane Proteins—Vectorial labeling with sulfonated succimidyl ester derivatives of biotin (42Hastie C. Saxton M. Akpan A. Cramer R. Masters J.R. Naaby-Hansen S. Oncogene. 2005; 24: 5905-5913Crossref PubMed Scopus (44) Google Scholar) was performed prior to interferon exposure or at the end of each treatment period. For avidin affinity chromatography, sulfo-NHS-SS-biotin-labeled cells were lysed in 1% octyl-β-d-glucopyranoside (OGP) in PBS, containing a protease inhibitor mixture with or without EDTA (Complete, Roche Applied Science). Where appropriate, OGP extractions were performed in the presence of the following phosphatase inhibitors: 1 μm okadaic acid, 5 μm α-cyano-3-phenoxybenzyl α-(4-chlorophenyl) isovalerate, and 5 μm potassium bisperoxo(1,10-phenanthroline) oxovanadate. Protein concentrations were determined by a modified Lowry assay (RC DC protein assay, Bio-Rad). The OGP-solubilized SS-biotin-labeled membrane fractions were purified by avidin affinity chromatography as described previously (42Hastie C. Saxton M. Akpan A. Cramer R. Masters J.R. Naaby-Hansen S. Oncogene. 2005; 24: 5905-5913Crossref PubMed Scopus (44) Google Scholar). The eluted fractions were dialyzed against distilled water, vacuum-concentrated, rehydrated in electrophoresis buffer, the isolated membrane proteins separated by SDS-PAGE or two-dimensional gel electrophoresis, and visualized by Coomassie or silver staining. In-gel digestion and mass spectrometry analysis of the generated peptide mixtures were performed as described previously (45Naaby-Hansen S. Nagano K. Gaffney P. Masters J. Cramer R. Methods Mol. Med. 2003; 81: 277-297PubMed Google Scholar). In some experiments the enriched surface fractions were transferred to NC membranes following electrophoresis and probed with horseradish peroxidase-avidin or antibodies. Avidin- and antibody binding was detected by the enhanced chemiluminescence technique. Secondary antibody-alone controls were included and were negative in all cases. High salt or chelating dissociation buffers were used to release peripherally attached proteins from the surface of intact cells. The cells were washed twice in PBS followed by a phosphate buffer with protease inhibitors and either CaCl2 (0.5-15 mm) or EDTA (0.5-5 mm), and surface proteins were released by gentle swirling. Calcium- and EDTA-free buffer treatments served as control. After 2 min the dissociation buffer was removed by aspiration, and the released proteins were concentrated by spin-vac centrifugation before being separated by SDS-PAGE, transferred to blotting membranes, and stained with antibody. Alternatively, peripherally attached proteins were released from biotinylated cells by treatment with a hypertonic phosphate buffer containing 0.65 m NaCl and protease inhibitors, for 5 min at room temperature. The high salt buffer was removed by aspiration and diluted to isoosmolarity, and avidin-conjugated agarose beads (20 μl/ml) were added. After 15 min of mixing by rotation, proteins bound to the avidin beads were isolated by centrifugation. Following five washes in isotonic PBS, pH 7.4, plus protease inhibitors, the purified proteins were eluted from the beads by heating in a reducing Laemmli buffer and separated by SDS-PAGE. Analysis of ABCA-1 Phosphorylation—[33P]Orthophosphate labeling was employed to investigate the phosphorylation status of ABCA1 in IFNγ-treated cells. Cells were grown to 70% confluence, washed twice in phosphate-free Dulbecco's modified Eagle's medium containing 0.1% fetal bovine serum, incubated for 1 h in the same medium, and labeled with 0.1 mCi/ml [33P]orthophosphate for 6 h prior to exposure to interferon. After different stimulation periods, the cells were lysed in RIPA buffer containing 1% Nonidet P-40 and 0.2% deoxycholate in PBS, pH 7.4, plus the protease and phosphatase inhibitor mixtures. ABCA1 and associated binding partners were then isolated by immunoprecipitation with monospecific, purified rabbit antibody (Novus Biologicals NB 400-105). Following SDS-PAGE separation, the immunopurified radiolabeled proteins were transferred onto polyvinylidene difluoride membranes, dried, and visualized by PhosphorImaging on a Molecular Imager FX scanner (Bio-Rad). Live Cell Immunofluorescence Staining—Live cell immunofluorescence was performed using the protocol of Groves (46Groves, A. (1992) DNA Amplification in Mammalian Cells, Ph.D. thesis, University College London, UKGoogle Scholar). Live cells were initially incubated with primary antibodies against annexin 2 and p11 and with cholera toxin. The cells were then fixed, permeabilized, and incubated with phalloidin to visualize the actin cortex, and finally stained with fluorophore-conjugated secondary antibodies. Cell Invasion Assays—The Biocoat Matrigel invasion chamber system (BD Biosciences) was used to assay the invasive potential of cells cultured in the presence or absence of IFNγ, employing the protocol of Falcone et al. (25Falcone D.J. Borth W. Khan K.M. Hajjar K.A. Blood. 2001; 97: 777-784Crossref PubMed Scopus (90) Google Scholar). To evaluate the effect of antibody blockage, the Matrigel was re-hydrated in normal medium or in a medium containing 25 μg/ml mouse monoclonal antibody against annexin 2 (catalog number 034400, Zymed Laboratories Inc.) at 4 °C. The same concentration of an isotypic mouse monoclonal anti-rabbit IgG antibody (Amersham Biosciences) was used as control. A similar protocol was used to assay the invasive effects of annexin 2-GFP transfection, with the exceptions that the cells here were obtained from a 24-well plate, and the migrants were counted using a fluorescence microscope. To study the effect instigated by inhibition of calpain, 1542CP3TX cells were pretreated for 3 h with 30 μm type-II calpain inhibitor (Calbiochem) or with a control solution of 0.01% Me2SO. The Biocoat Matrigel wells were rehydrated in media containing either Me2SO, IFNγ + calpain inhibitor + Me2SO, calpain inhibitor + Me2SO, or IFNγ + Me2SO, to ensure that seeded cells remained in the appropriate stimulant conditions during subsequent phases of the assay. After 22 h of incubation, the cells were fixed with 4% paraformaldehyde and stained with Hoescht. Migrants were quantified by fluorescence microscopy. Transfection of LNCaP Cells with a GFP-Annexin 2 Fusion Construct—LNCaP cells were transfected with a plasmid encoding an annexin 2-GFP fusion protein or with the GFP vector alone as a control, to examine how expression of annexin 2 affected their invasive capacity. A pEGFP-C1 vector-based wild type annexin 2 construct (47Merrifield C.J. Rescher U. Almers W. Proust J. Gerke V. Sechi A.S. Moss S.E. Curr. Biol. 2001; 11: 1136-1141Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) was transfected into LNCaP cells using the FuGENE transfection reagent (Roche Applied Science) twice at 18-h intervals, and cultures demonstrating high GFP and GFP-annexin 2 expression after 48 h were used in the Matrigel invasion assay described above. IFNγ Reduces Cell Surface Expression of Annexin 2—Vectorial labeling with sulfo-N-hydroxysuccinimidyl derivatives of biotin was combined with two-dimensional gel electrophoresis and avidin affinity blotting to study the effect of IFNγ treatment on surface composition in 1542CP3TX prostate cancer cells. IFNγ increased the number of biotinylated proteins and enhanced the membrane density of several surface proteins (Fig. 1A). Relatively few protein spots were down-regulated by IFNγ treatment (upward arrows in Fig. 1A), some because of altered abundance of the individual isoforms in charge trains (white rectangles in Fig. 1A). To identify proteins regulated by IFNγ and achieve optimal representation of hydrophobic and high molecular weight membrane proteins, avidin affinity-purified surface proteins were separated by SDS-PAGE prior to analysis by mass spectrometry (42Hastie C. Saxton M. Akpan A. Cramer R. Masters J.R. Naaby-Hansen S. Oncogene. 2005; 24: 5905-5913Crossref PubMed Scopus (44) Google Scholar). Although IFNγ increased both the repertoire and abundance of proteins in the avidin-isolated biotinylated membrane fractions, the surface density of an abundant 36-kDa protein was strongly reduced after 72 h of exposure to the cytokine (oblique arrows in Fig. 1B). The protein was identified as annexin 2 by matrix-assisted laser desorption/ionization-mass spectrometry analysis of peptides generated by trypsinization of the excised Coomassie-stained gel band (Fig. 1C). Densitometry analysis of gel images from three independent experiments revealed that the surface expression of the 36-kDa annexin 2 band was reduced by 4-fold after 72 h of IFNγ treatment. Immunoblotting of avidin-purified biotinylated membrane fractions from treated and untreated 1542CP3TX cells confirmed the identity of the 36-kDa band and the reduction in annexin 2 surface expression induced by IFNγ (inset in Fig. 1C). A 54-kDa protein that was strongly up-regulated in both whole cell extracts and membrane fractions from IFNγ-treated cells (horizontal arrow in Fig. 1B) was identified as interferon-induced protein 53 (IFP53) by mass spectrometry (data not shown). Annexin 2 was readily released from the cell surface by treatment with chelating or high salt dissociation buffers, suggesting that the protein is peripherally bound to the plasma membrane in a calcium-dependent manner (supplemental Fig. 1). To study the kinetics of annexin 2 down-regulation, EDTA-released membrane fractions were obtained at various times after addition of IFNγ and analyzed by immunoblotting. Although the membrane density of annexin 2 remained high during the first 2hofIFNγ treatment, it was strongly reduced after 4 h (Fig. 1D). Following6hof exposure to IFNγ, annexin 2 was barely detectable in EDTA-released surface fractions from 1542CP3TX cells (Fig. 1D and Fig. 4H). Long term IFNγ treatment did not alter the abundance of annexin 2 in whole cell detergent extracts from 1542CP3TX cells (Fig. 1E). The global expression levels of annexin 2 in the benign prostate epithelial cell line PRE 2.8 and in the prostate cancer cell lines PC3 and DU145 were likewise unaffected by cytokine treatment (supplemental Fig. 2). In accordance with previous findings (48Chetcuti A. Margan S.H. Russell P. Mann S. Millar D.S. Clark S.J. Rogers J. Handelsman D.J. Dong Q. Cancer Res. 2001; 61: 6331-6334PubMed Google Scholar), annexin 2 was not detected in LNCaP prostate cancer cells (supplemental Fig. 2). Taken together, these findings show that IFNγ induces a significant reduction in the membrane density of annexin 2 in 1542CP3TX prostate cancer cells, and that the suppressive effect is specific for peripherally attached surface proteins. IFNγ Alters the Surface Localization of Annexin 2—The effects of IFNγ were next analyzed by surface staining of intact, viable cells. Cholera toxin was used to stain GM1 gangliosides, which are present in high abundance at lipid rafts, and phalloidin was used to stain F-actin after membrane permeabilization. Immunofluorescence staining with an antibody against annexin 2 confirmed that the protein is present on the surface of 1542CP3TX cells and that 24 h of treatment with IFNγ strongly reduces the membrane density of annexin 2 (Fig. 2A). In addition to the reduction in peripheral expression, IFNγ altered the distribution of annexin 2 on the cell surface. Whereas a generalized pattern of annexin 2 staining was observed over the surface of unstimulated cells, IFNγ treatment concentrated the remaining annexin 2 molecules to lipid rafts (Fig. 2A). Whereas the intensity of p11 surface staining also was strongly reduced following 24 h of IFNγ treatment, the localization of p11 remained unaltered closely mirroring that of the cholera toxin-positive lipid raft structures in both treated and untreated cells. 24 h exposure to IFNγ did not reduce the global expression level of p11 (supplemental Fig. 3), indicating that the suppressive effect of the cytokine again is specific for proteins expressed on the cell surface. One possible interpretation of these observations is that annexin 2 exists both as a monomer and in heterotetrameric complex with p11 on the surface of 1542CP3TX cells, and although the monomer binds to membrane lipids outside raft areas and is more susceptible to IFNγ-induced down-regulation, the heterotetramer is situated within the lipid rafts. IFNγ Suppresses the Invasive Capacity of Prostate Cancer Cells—To investigate whether IFNγ treatment affects the invasiveness of prostate cancer cells, we compared 1542CP3TX and LNCaP cells in a Biocoat Matrigel invasion chamber assay. LNCaP cells do not express annexin 2 as a result of promoter silencing by hypermethylation (48Chetcuti A. Margan S.H. Russell P. Mann S. Millar D.S. Clark S.J. Rogers J. Handelsman D.J. Dong Q. Cancer Res. 2001; 61: 6331-6334PubMed Google Scholar) (supplemental Fig. 2). Invasion was significantly reduced (p = 0.029) in 1542CP3TX cells after interferon treatment, but LNCaP cells were unaffected (Fig. 3A). Exposure to an antibody against annexin 2 significantly reduced the number of transmigrating 1542CP3TX cells (Fig. 3B), whereas a similar concentration of mouse IgG, raised against rabbit immunoglobulin, had no effect (Fig. 3C). To confirm that annexin 2 expression influences the invasiveness of prostate cancer cells, annexin 2 was expressed in LNCaP cells as a GFP fusion protein. Although the annexin 2-GFP fusion protein was most abundant in the nucleus and cytosol, a peripheral staining pattern indicative of membrane-localized proteins was seen in most of the transfected cells (supplemental Fig. 4). A graphic representation of data from six independent experiments is shown in Fig. 3D, demonstrating a significant increase (p < 0.002) in the invasive capacity of LNCaP cells transfected with annexin 2 compared with empty vector alone. Consistent with the heterotetrameric anx2t complex function as an anchorage and activation site for procathepsin B and plasminogen on cell surfaces (37Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. Chem. 2000; 275: 12806-12812Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 25Falcone D.J. Borth W. Khan K.M. Hajjar K.A. Blood. 2001; 97: 777-784Crossref PubMed Scopus (90) Google Scholar, 30Mai J. Waisman D.M. Sloane B.F. Biochim. Biophys. Acta. 2000; 1477:" @default.
• W2006733013 created "2016-06-24" @default.
• W2006733013 creator A5019178495 @default.
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• W2006733013 title "Interferon-γ Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells" @default.
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• W2006733013 doi "https://doi.org/10.1074/jbc.m800189200" @default.
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