Expression of PTRF in PC-3 Cells Modulates Cholesterol Dynamics and the Actin Cytoskeleton Impacting Secretion Pathways
2011; Elsevier BV; Volume: 11; Issue: 2 Linguagem: Inglês
10.1074/mcp.m111.012245
ISSN1535-9484
AutoresKerry L. Inder, Yu Zi Zheng, Melissa J. Davis, Hyeongsun Moon, Dorothy Loo, Hien D. Nguyen, Judith A. Clements, Robert G. Parton, Leonard J. Foster, Michelle M. Hill,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoExpression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression. Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression. Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer related deaths in developed countries. Although localized prostate cancer is treatable, metastatic recurrence, together with development of androgen-independence, leads to advanced prostate cancer, which currently has a low survival rate. Caveolin-1, a cholesterol-binding integral membrane protein, has been shown to be up-regulated in prostate cancer metastasis and is associated with androgen-independence and aggressive recurrence of the disease (1Bennett N. Hooper J.D. Lee C.S. Gobe G.C. Androgen receptor and caveolin-1 in prostate cancer.IUBMB Life. 2009; 61: 961-970Crossref PubMed Scopus (24) Google Scholar). A prostate cancer mouse model showed that genetic ablation of caveolin-1 delays the onset of advanced prostate cancer (2Williams T.M. Hassan G.S. Li J. Cohen A.W. Medina F. Frank P.G. Pestell R.G. Di Vizio D. Loda M. Lisanti M.P. Caveolin-1 promotes tumor progression in an autochthonous mouse model of prostate cancer: genetic ablation of Cav-1 delays advanced prostate tumor development in tramp mice.J. Biol. Chem. 2005; 280: 25134-25145Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), underscoring its importance in prostate cancer progression. Hence understanding caveolin-1 action in prostate cancer progression is crucial for the design of novel intervention strategies to manage this devastating disease. Caveolin-1 is a major structural component of caveolae, specialized lipid raft microdomains of the plasma membrane characterized by their flask shaped invaginations (3Parton R.G. Simons K. The multiple faces of caveolae.Nat. Rev. Mol. Cell Biol. 2007; 8: 185-194Crossref PubMed Scopus (1155) Google Scholar). Lipid rafts and caveolae are thought to participate in a variety of cellular processes including lipid regulation, endocytosis, cell adhesion, and signal transduction (4Parton R.G. Richards A.A. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms.Traffic. 2003; 4: 724-738Crossref PubMed Scopus (494) Google Scholar). However, a clear delineation between lipid rafts and caveolae function remains to be made. Recently, it was revealed that the protein Polymerase I and transcript release factor (PTRF) 1The abbreviations used are:PTRFpolymerase I and transcript release factorDRMsdetergent-resistant membranesLC-MS/MSliquid chromatography-tandem mass spectrometrySILACstable isotope labeling by amino acids in cell cultureMβCDmethyl-β-cyclodextrinIL-6Interleukin-6KLK-6Kallikrein 6PBSphosphate-buffered saline. 1The abbreviations used are:PTRFpolymerase I and transcript release factorDRMsdetergent-resistant membranesLC-MS/MSliquid chromatography-tandem mass spectrometrySILACstable isotope labeling by amino acids in cell cultureMβCDmethyl-β-cyclodextrinIL-6Interleukin-6KLK-6Kallikrein 6PBSphosphate-buffered saline., also known as cavin-1, is an essential cofactor required for stabilization of caveolae at the plasma membrane (5Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 6Liu L. Brown D. McKee M. Lebrasseur N.K. Yang D. Albrecht K.H. Ravid K. Pilch P.F. Deletion of Cavin/PTRF causes global loss of caveolae, dyslipidemia, and glucose intolerance.Cell Metab. 2008; 8: 310-317Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The loss of PTRF results in the loss of caveolae, as well as a decrease in caveolin-1 stability (5Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). These results demonstrate a crucial role for PTRF in caveolae structure and function. Given these new insights, the role of PTRF in relation to its function in caveolae is only beginning to be examined in prostate cancer (7Gould M.L. Williams G. Nicholson H.D. Changes in caveolae, caveolin, and polymerase 1 and transcript release factor (PTRF) expression in prostate cancer progression.Prostate. 2010; 70: 1609-1621Crossref PubMed Scopus (43) Google Scholar). polymerase I and transcript release factor detergent-resistant membranes liquid chromatography-tandem mass spectrometry stable isotope labeling by amino acids in cell culture methyl-β-cyclodextrin Interleukin-6 Kallikrein 6 phosphate-buffered saline. polymerase I and transcript release factor detergent-resistant membranes liquid chromatography-tandem mass spectrometry stable isotope labeling by amino acids in cell culture methyl-β-cyclodextrin Interleukin-6 Kallikrein 6 phosphate-buffered saline. Intriguingly, although caveolin-1 is predominately a membrane protein, aggressive prostate cancer cell lines have been shown to secrete biologically active caveolin-1 (8Tahir S.A. Yang G. Ebara S. Timme T.L. Satoh T. Li L. Goltsov A. Ittmann M. Morrisett J.D. Thompson T.C. Secreted caveolin-1 stimulates cell survival/clonal growth and contributes to metastasis in androgen-insensitive prostate cancer.Cancer Res. 2001; 61: 3882-3885PubMed Google Scholar). Furthermore, secreted caveolin-1 stimulates cell growth and angiogenesis in tumor models (8Tahir S.A. Yang G. Ebara S. Timme T.L. Satoh T. Li L. Goltsov A. Ittmann M. Morrisett J.D. Thompson T.C. Secreted caveolin-1 stimulates cell survival/clonal growth and contributes to metastasis in androgen-insensitive prostate cancer.Cancer Res. 2001; 61: 3882-3885PubMed Google Scholar, 9Tahir S.A. Yang G. Goltsov A.A. Watanabe M. Tabata K. Addai J. Fattah el M.A. Kadmon D. Thompson T.C. Tumor cell-secreted caveolin-1 has proangiogenic activities in prostate cancer.Cancer Res. 2008; 68: 731-739Crossref PubMed Scopus (72) Google Scholar). Serum caveolin-1 has been detected in recurrent prostate cancer after radical prostatectomy and has been proposed to be a marker for disease recurrence (8Tahir S.A. Yang G. Ebara S. Timme T.L. Satoh T. Li L. Goltsov A. Ittmann M. Morrisett J.D. Thompson T.C. Secreted caveolin-1 stimulates cell survival/clonal growth and contributes to metastasis in androgen-insensitive prostate cancer.Cancer Res. 2001; 61: 3882-3885PubMed Google Scholar, 10Tahir S.A. Ren C. Timme T.L. Gdor Y. Hoogeveen R. Morrisett J.D. Frolov A. Ayala G. Wheeler T.M. Thompson T.C. Development of an immunoassay for serum caveolin-1: a novel biomarker for prostate cancer.Clin. Cancer Res. 2003; 9: 3653-3659PubMed Google Scholar). In addition to classical secretion, prostate cells also secrete proteins via membranous storage vesicles referred to as prostasomes. It is thought that prostasomes are formed in a similar manner to exosomes, that is, via multivesicular bodies that have encapsulated cytoplasmic proteins (11Théry C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat. Rev. Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3741) Google Scholar). Prostate epithelial cells secrete prostasomes into the prostate fluid where they have important functions related to fertility. More recently it has become apparent that prostate cancer cell lines also secrete prostasomes and it is thought they may act to modulate the local tumor environment (12Jansen F.H. Krijgsveld J. van Rijswijk A. van den Bemd G.J. van den Berg M.S. van Weerden W.M. Willemsen R. Dekker L.J. Luider T.M. Jenster G. Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.Mol. Cell. Proteomics. 2009; 8: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Llorente A. de Marco M.C. Alonso M.A. Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line.J. Cell Sci. 2004; 117: 5343-5351Crossref PubMed Scopus (84) Google Scholar, 14Llorente A. van Deurs B. Sandvig K. Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells.Eur. J. Cell. Biol. 2007; 86: 405-415Crossref PubMed Scopus (58) Google Scholar). Prostasome secretion is modulated by cholesterol levels and caveolin-1 has been identified as a component of prostasomes in the human prostate carcinoma PC-3 cell line (13Llorente A. de Marco M.C. Alonso M.A. Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line.J. Cell Sci. 2004; 117: 5343-5351Crossref PubMed Scopus (84) Google Scholar). These data have prompted us to examine the role of caveolin-1 and PTRF in secretion of prostate cancer cells. We have previously shown that PC-3 cells express abundant caveolin-1 but lack PTRF, and that ectopic expression of PTRF in PC-3 cells results in caveolae formation (5Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). Furthermore, PTRF expression in PC-3 cells resulted in reduced directional migration, partly because of attenuation of secreted MMP9 (15Aung C.S. Hill M.M. Bastiani M. Parton R.G. Parat M.O. PTRF-cavin-1 expression decreases the migration of PC3 prostate cancer cells: role of matrix metalloprotease 9.Eur J. Cell Biol. 2011; 90: 136-142Crossref PubMed Scopus (58) Google Scholar). Given that caveolin-1 and caveolae participate in a range of cellular processes, we undertook quantitative subcellular proteomics analysis using our established PC-3 cell lines, which express GFP-PTRF or GFP (5Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar), in order to acquire a global analysis of changes induced by PTRF expression. Systems analysis of the subcellular proteomes points to a role for PTRF in modulating cholesterol dynamics and regulation of the actin cytoskeleton, impacting the trafficking of multiple secretion pathways. Importantly, PTRF reduced the secretion of a specific subset of proteins including secreted proteases, cytokines, and growth factors. Our study reveals the potential for PTRF as a therapeutic option that could target a spectrum of secreted factors rather than a single molecule. The following materials were obtained from the indicated commercial sources: Roswell Park Memorial Institute (RPMI)-1640 medium, l-glutamine, Geneticin (G418), and cell culture trypsin were from Invitrogen (Carlsbad, CA); Fetal bovine serum was from Bovogen (Melbourne, Australia); l-lysine, l-arginine, and l-leucine deficient RPMI 1640, l-leucine, Triton X-100, sodium deoxycholate, 2-(N-morpholino)ethanesulfonic acid, dithiotheitol, iodoacetamide, filipin III, water-soluble cholesterol, protease inhibitors, and antibodies against IL-6 and β-actin were from Sigma (St. Louis, MO); 13C615N2-lysine and 13C615N4-arginine were from Silantes (Munchen, Germany); Sequencing grade modified porcine trypsin was from Promega (Madison, WI); Antibody against kallikrein 6 was purchased from Santa Cruz (Santa Cruz, CA); Antibodies against caveolin-1 and flotillin were from BD Biosciences; Antibody against filamin A was from Millipore (Billerica, MA); PTRF antibodies were produced by immunizing rabbits with a synthetic peptide corresponding to the C-terminal 12 amino acids of mouse PTRF, and affinity purified from serum (16Bastiani M. Liu L. Hill M.M. Jedrychowski M.P. Nixon S.J. Lo H.P. Abankwa D. Luetterforst R. Fernandez-Rojo M. Breen M.R. Gygi S.P. Vinten J. Walser P.J. North K.N. Hancock J.F. Pilch P.F. Parton R.G. MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes.J. Cell Biol. 2009; 185: 1259-1273Crossref PubMed Scopus (207) Google Scholar). GFP-PTRF has been described previously (5Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). PC-3, PC-3 GFP, and PC-3 GFP-PTRF stable cells were grown and maintained in RPMI 1640 medium containing 10% fetal bovine serum. Stable cell lines were maintained in 0.1 mg/ml G418. For SILAC experiments, GFP and GFP-PTRF PC-3 cells were maintained in media lacking Lysine and Arginine with dialyzed FBS and supplemented with the following amino acids: "0/0 " for the normal isotopic Lys and Arg and "8/10 " for 13C615N2-Lys and 13C615N4-Arg. Cell populations were amplified 200-fold in the labeling media to achieve >99% incorporation as confirmed by liquid chromatography-tandem MS (LC-MS/MS). Where required, cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For each analysis, two to three 15-cm plates of PC-3 cells were used per condition for total membrane, detergent-resistant membrane extractions, secreted proteome and prostasome extraction. Briefly, PC-3 cells grown to 60 to 70% confluency were washed five times with phosphate-buffered saline (PBS) and then incubated for 24 h in serum free media. Any cell debris was removed by centrifugation at 600 × g for 10 min at 4 °C. Cleared cell culture supernatant was concentrated through an Amicon 10 kDa cutoff spin column and protein concentration was measured using BCA assay. Equal protein levels of secretome prepared from the two cell lines were mixed for isolation of prostasomes or in-solution digest. Isolation of prostasomes from cleared conditioned media involved sequential centrifugation; 10,000 × g 30 min 4 °C spin to remove any cell fragments not pelleted at 600 × g, then centrifugation of the supernatant at 100,000 × g, 2 h, 4 °C. Prostasome pellets were washed with PBS and again pelleted at 100,000 × g, 2 h, 4 °C. Pellets were resuspended in 1% sodium deoxycholate. The P100 fraction was isolated from cells as previously described (17Inder K.L. Lau C. Loo D. Chaudhary N. Goodall A. Martin S. Jones A. van der Hoeven D. Parton R.G. Hill M.M. Hancock J.F. Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction.J. Biol. Chem. 2009; 284: 28410-28419Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Detergent resistant membranes (DRMs) were prepared by solubilizing cells in lysis buffer (1% Triton X-100, 25 mm 2-(N-morpholino) ethanesulfonate pH 6.5, 150 mm NaCl, (2-(N-morpholino) ethanesulfonate -buffered saline or MBS) plus protease inhibitors) and incubated on ice for 30 min. Relative protein concentrations were determined using Bradford assay and equal protein levels from each SILAC condition were mixed together. Lysates were adjusted to 40% sucrose by addition of an equal volume of 80% sucrose (in MBS), followed by successive layers of 30 and 5% sucrose. The gradients were centrifuged for 18 h at 160,000 × g, 4 °C and the white, light-scattering band appearing between 30 and 5% sucrose collected. The extracted layer was diluted out ∼threefold with MBS and pelleted by centrifugation at 160,000 × g for 2 h, 4 °C. All steps above were carried out at 4 °C. Samples were resuspended or diluted with 1% sodium deoxycholate in 50 mm NH4HCO3 and heat solubilized for 10 min at 95 °C. Fifty micrograms of sample was reduced with 1 μg dithiotreitol, alkylated with 5 μg iodoacetamide and trypsin digested. An equal volume of 3% acetonitrile, 1% trifluoroacetic acid, 0.5% acetic acid was added to acidify the sample. In some experiments, digested peptides were also further fractionated by strong cation exchange chromatography into five fractions using 0, 20, 50, 100, and 500 mm NH4CH3COO. Samples (4 μg) were analyzed using a 1200 Series nano HPLC and Chip-Cube Q-TOF 6510 (Agilent Technologies). Peptides were resolved by 160 nl (75 mm * 150 μm) high capacity C18 reverse phase chip with 55 min gradient from 0 to 45% acetonitrile with the Vcap 1850 V, fragmentor 175 V. Precursor ions were selected in the range of 100–3200 m/z and fragment ions at 59–3200 m/z; reference ion mix was applied. SILAC samples were analyzed in auto MS/MS mode, with 8 MS and 4 MS/MS per second. Raw MS data are available at PRIDE, accession numbers 19891–19906. Mass spectra extraction, database searching, and quantitative ratios were performed using Spectrum Mill software (Agilent, A03.03) against Swissprot Human database (release-2010_03 containing 23,000 entries). Cysteine carbamidomethylation and SILAC amino acids N-Lys, 13C615N2-Lys, N-Arg, and 13C615N4-Arg were used as a fixed/mix modifications as appropriate and oxidized methionine was selected as variable modification. Other parameters include up to 2 miss cleavages for trypsin; ±20 ppm and ±50 ppm accuracy for MS and MS/MS measurements respectively. Positive identification required a protein score >11, peptide score >10, and >60% scored peak intensity. Single peptide identifications were excluded from further analysis. Mean SILAC ratio and standard deviation was calculated using all the peptide ratios matched to a protein, and p values were calculated using the peptide SILAC ratios. False Discovery Rates were calculated by dividing the number of false positives (identified by searching against a reverse decoy database (18Reidegeld K.A. Eisenacher M. Kohl M. Chamrad D. Körting G. Blüggel M. Meyer H.E. Stephan C. An easy-to-use Decoy Database Builder software tool, implementing different decoy strategies for false discovery rate calculation in automated MS/MS protein identifications.Proteomics. 2008; 8: 1129-1137Crossref PubMed Scopus (68) Google Scholar)), by the total number of proteins identified. For each replicate the false discovery rate was <1.25%. Proteins with a SILAC p value < 0.05 were submitted to GeneGo for identification of Gene Ontology (GO) terms over represented in each list. Correction for multiple hypothesis testing was performed by controlling for the false discovery rate at p = 0.05. GeneGo networks were built using the shortest path and two connections algorithm. Low trust interactions and unconnected nodes were removed. Samples were resolved on SDS-PAGE gels and transferred to polyvinylidene difluoride using wet transfer. The membranes were blocked in 5% milk powder and then probed with primary antibody for 1 h. After washing, the membrane was incubated with the appropriate horseradish peroxidase-coupled secondary antibody for 1 h and then developed using SuperSignal West Pico chemiluminescence and captured on film (Kodak). Cells were grown on coverslips, transiently transfected and fixed in 4% paraformaldehyde after 24 h. For cholesterol staining, the coverslip was incubated with 50 μg/ml filipin for 2 h in the dark. Coverslips were washed three times with PBS, mounted, and visualized. For all other staining the coverslips were fixed in 4% paraformaldehyde, then permeabilized and blocked in 0.1% Triton-X, 1% BSA in PBS for 30 min. Anti-caveolin-1, anti-filamin A, or Texas Red conjugated phalloidin was incubated on the coverslips for 1 h. Where necessary, after washing in PBS, the appropriate secondary antibody was incubated for another hour. Coverslips were washed in PBS, mounted using Vectashield and visualized using a Zeiss Meta 510 confocal microscope. Cholesterol assay was performed using the Amplex Red cholesterol assay kit (Invitrogen) according to the manufacturer's instructions. To gain a comprehensive view of cellular changes induced upon PTRF expression, we used SILAC with subcellular fractionation to quantitatively analyze the effect of PTRF expression on the PC-3 subproteomes. Based on the potential caveolar role of PTRF, we focused on four fractions: secreted proteins, secreted vesicles (prostasomes), total membrane fraction prepared as a pellet after 100,000 × g spin (P100), or DRM, a fraction enriched in lipid raft microdomains (Fig. 1A). Equal amount of total cell lysate from SILAC-labeled PC-3 cells stably expressing GFP (no PTRF expression) or GFP-PTRF (PTRF mainly localized to caveolae) (data not shown) were combined. The P100 or the DRM fractions were isolated and then analyzed by mass spectrometry (Fig. 1A). The secretome was collected by growing cells in serum free media for 24 h and using equal protein amount of conditioned media for each cell type. No significant difference was observed in the viability of cells grown in serum free media (93.12 ± 7.62%) compared with cells grown in 10% serum containing media (100%). The vesicular component of the secretome (also termed prostasomes), was further fractionated from the secretome and then both fractions were analyzed by mass spectrometry (Fig. 1A). An overall mean was calculated for each protein using SILAC ratios for peptides from the four independent measurements (supplemental Tables S1 and S3) and then analyzed for proteins significantly changed (p value ≤ 0.05) upon PTRF expression. We found that PTRF expression significantly decreased 136 (25%) from the 544 proteins identified in the P100 fraction (Fig. 1B). In the DRM fraction, 103 proteins (29%) were decreased with PTRF expression, from a total of 358 proteins quantified (Fig. 1B). Very few proteins were increased in abundance in the P100 or DRM fractions, five and six respectively (Fig. 1B). PTRF expression also caused a significant reduction in proteins from the secreted and prostasome fractions: 114 proteins (31%) were secreted less with PTRF expression from a total of 370 proteins identified, and 112 proteins (30%) decreased from a total of 367 proteins in the prostasome (Fig. 1B). In contrast, very few proteins were increased in the secretome and prostasome, nine and 11 respectively (Fig. 1B). Importantly PTRF expression did not alter the total protein content of any of the fractions (Fig. 1C). These results reveal a specific change in the membrane targeting and/or secretion of a subset of proteins upon PTRF expression in PC-3 cells. Previously we reported PTRF expression reduced PC-3 cell migration though collagen-coated Boyden chamber partly via reduction of MMP9 secretion (15Aung C.S. Hill M.M. Bastiani M. Parton R.G. Parat M.O. PTRF-cavin-1 expression decreases the migration of PC3 prostate cancer cells: role of matrix metalloprotease 9.Eur J. Cell Biol. 2011; 90: 136-142Crossref PubMed Scopus (58) Google Scholar). Because cell migration and invasion requires co-ordination between cellular events and secreted proteases, we examined the quantitative proteomics data for proteins with a role in cancer and cell migration. Pathways enrichment analysis using GeneGo software revealed that PTRF expression most significantly reduces the secretion of proteins involved in cell adhesion and cytoskeleton remodeling (Fig. 2A). Interestingly, PTRF modulated similar pathways in the prostasome fraction. Because the prostasome is a subfraction of the secretome, one possible explanation for the overlap is that PTRF expression specifically attenuated the secretion of vesicles. To examine this possibility, we analyzed the proteins common to the secretome and prostasome fraction (Fig. 2B). The result shows that 244 proteins (66%) were identified in both fractions, indicating that prostasomes contribute a significant portion of total secreted proteins. In the subset of proteins significantly changed by PTRF expression, 61 (∼50%) were common to the secretome and prostasome, meaning that the reduction in secreted proteins upon PTRF expression could only be partially explained by a reduction in prostasomal proteins. Closer inspection of the proteins reduced in both the secretome and prostasome fractions by PTRF expression identified a number of proteins with established links to cancer and in particular metastasis, including extracellular matrix proteins, secreted proteases, and cytokines (Table I). We selected two proteins that have been previously implicated in prostate cancer (19Nakashima J. Tachibana M. Horiguchi Y. Oya M. Ohigashi T. Asakura H. Murai M. Serum interleukin 6 as a prognostic factor in patients with prostate cancer.Clin. Cancer Res. 2000; 6: 2702-2706PubMed Google Scholar, 20Twillie D.A. Eisenberger M.A. Carducci M.A. Hseih W.S. Kim W.Y. Simons J.W. Interleukin-6: a candidate mediator of human prostate cancer morbidity.Urology. 1995; 45: 542-549Abstract Full Text PDF PubMed Scopus (269) Google Scholar, 21Sardana G. Marshall J. Diamandis E.P. Discovery of candidate tumor markers for prostate cancer via proteomic analysis of cell culture-conditioned medium.Clin. Chem. 2007; 53: 429-437Crossref PubMed Scopus (67) Google Scholar) for verification by Western blotting, namely kallikrein 6 (KLK6) and interleukin-6 (IL-6). To cross-validate the stable cell line quantitative proteomics results, we chose transient transfection of wild-type PC-3 cells for these experiments. Conditioned media collected from PC-3 cells transiently transfected with control GFP or GFP-PTRF plasmids were immunoblotted with antibodies against KLK-6 and IL-6. This result confirmed that expression of PTRF reduced secreted KLK-6 and IL-6 (Fig. 2C, left panel). Whole cell lysates of GFP and GFP-PTRF cells showed no change in expression levels (Fig. 2C, right panel) indicating that PTRF expression does not affect total cellular levels of KLK-6 and IL-6 but rather specifically modulates their secretion.Table IList of selected proteins that were reduced in the secretome and prostasome fraction of SILAC PC-3 cells expressing PTRF. Protein level
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