Differential protein synthesis and expression levels in normal ... - Nature

Oncogene (2004) 23, 1693–1703

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Differential protein synthesis and expression levels in normal and neoplastic human prostate cells and their regulation by type I and II interferons Kohji Nagano1,4, John R Masters2, Akunna Akpan1, Alice Yang1, Steve Corless1, Chris Wood1, Claire Hastie1, Marketa Zvelebil1,3, Rainer Cramer1,3, and Soren Naaby-Hansen*,1,3 1 Ludwig Institute for Cancer Research, Royal Free and University College London Medical School, London, UK; 2Prostate Cancer Research Center, Royal Free and University College London Medical School, London, UK; 3Department of Biochemistry and Molecular Biology, University College London, London, UK.

Protein expression and de novo synthesis in normal and prostate cancer cell lines derived from the same patient were compared by proteomic analysis, and the effects of INFa and INFc (INF ¼ interferon) determined. The expressions of several INF-inducible proteins, including MxA, Nmi, PA28a and IFP53, were downregulated in the cancer cells. INFc induced a more than twofold increase or decrease in the synthesis rates of almost twice as many proteins in the cancer cell line. The positive regulator of INF-induced transcription ISGF3c was upregulated in the cancer cells and inversely regulated by INFa and INFc in the normal and cancer cells. Moreover, ISGF3c’s induction by INFc in the cancer cells was more enhanced by simultaneous stimulation with EGF, than its induction in the normal cells. In all, 31 differentially regulated proteins were identified by mass spectrometry analysis, several of which are involved in chaperone-assisted protein folding in the endoplasmic reticulum (ER) or in regulated protein degradation. Our results suggest that the exclusion of proteins by the ER quality control system, crosstalk between the EGF- and INF-induced signalling pathways and the regulation of INF-inducible genes are all altered in the prostate cancer cells. The combination of upregulated activity in the growth-promoting PI3K/Akt pathway, suppression of Nmi and overexpression of hnRNP-K and c-myc proteins may explain why the prostate cancer cells were found to be more resistant to the growth inhibitory effects of INFc. Oncogene (2004) 23, 1693–1703. doi:10.1038/sj.onc.1207297 Published online 1 December 2003 Keywords: prostate cancer; interferon growth inhibitory effect of interferon

treatment;

*Correspondence: S Naaby-Hansen, Ludwig Institute for Cancer Research, Royal Free and University College London Medical School, Courtauld Building, 91 Riding House Street, London W1W 7BS, UK; E-mail: [email protected] 4 Current address: Department of Proteomics Research, Institute of Medical Sciences, University of Tokyo, Japan Received 29 July 2003; revised 26 September 2003; accepted 21 October 2003

Introduction Prostate cancer is the most frequently diagnosed tumor in men in Western industrialized countries and is among the most heterogeneous of cancers, both histologically and clinically (Isaacs et al., 2002). Early diagnosis provides an opportunity for curative surgery or radiotherapy. However, up to 30% of men undergoing radical prostatectomy relapse as a result of micrometastatic disease present at the time of surgery (Roberts et al., 2001). Advanced prostate cancer usually responds to androgen ablation for a median period of 12–18 months, but once hormone-resistant disease has developed there are currently no effective therapeutic options. Thus a major challenge is to identify the biological properties that drive tumor behavior and progression in prostate cancer, and to identify biomarkers that can be used to inform therapeutic choice for the individual patient. With the exception of prostate-specific antigen (PSA), no single protein has been shown to have sufficient prognostic or therapeutic utility to influence prostate cancer management (Singh et al., 2002). Downregulation of interferon (INF)-inducible genes appears to be associated with the development and progression of prostate cancer (Shou et al., 2002). Using an isogenic pair of transformed and nontransformed prostate epithelial cell lines, addition of IFN inhibited the proliferation of the transformed cells. In clinical samples, decreased expression of INF-inducible genes was seen in 30% of the 49 prostatic cancers examined (Shou et al., 2002). This study describes a two-dimensional (2D) gelbased proteomic analysis of protein synthesis rates and expression levels in a normal (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient (Bright et al., 1997). Metabolic labelling experiments revealed that the number of proteins both up- and downregulated in response to INFg treatment in the cancer cells was almost twice that in the normal cells, suggesting that INF-induced protein synthesis is deregulated in the prostate cancer cells. In addition, several proteins regulated by INFs, including GTPase MxA, N-myc interacting protein (Nmi), proteasome activator subunit 1 (PA28a), the chaperone APG-2 and tryptophanyltRNA synthetase (IFP53), were all downregulated in the cancer cells. INFg-specific downregulation of the

Functional proteomic analysis of prostate cancer cells K Nagano et al

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antigen-presenting surface chaperone gp96 in the cancer cells was also observed. The PI3K/Akt and the MAPK pathways were both upregulated in cancer cells compared to the normal cells. Our results provide support for the concept that deregulation of INF-induced gene activity may be involved in the pathogenesis of prostate cancer. The results also suggest that the cancer cell lines altered response to INFs and growth factors provides resistance to the growth inhibitory effects of INFg. Materials and methods Cell culture, metabolic labelling and preparation of whole-cell lysates The normal prostate epithelial cell line 1542-NP2TX and the prostate cancer cell line1542-CP3TX, derived from the same patient, were cultured in the presence of EGF as described by the originators (Bright et al., 1997). To examine the effect of INFs, 750 000 cells were plated per T80 flask and after 3 days culture, the medium was exchanged for fresh medium containing either or both IFN-a (500 U/ml) and IFN-g (500 U/ml) for 36 or 72 h prior to lysis. Protein synthesis was measured by labelling the cells with [35S]Met/Cys (0.2 mCi/ml of Promix; Amersham Pharmacia) for 18 h or during the last 18 h of cytokine treatment. For 2D gel electrophoresis (2DE), cells were lysed in a solution containing 4% CHAPS, 8 M urea, 2 M thiourea, 65 mM dithiothreitol (DTT), protease inhibitor cocktail with EDTA (Boehringer Mannheim) and phosphatase inhibitors (1 mM okadaic acid, 5 mM a-cyano-3-phenoxybenzyl a-(4chlorophenyl) isovalerate and 5 mM potassium bisperoxo (1,10-phenanthroline) oxovanadate). Protein concentrations were determined by Bradford assay (Coomassie Plus Protein Assay Reagent, Pierce) or by a modified Lowry assay (DC protein assay, Biorad). 2D gel electrophoresis Analytical isoelectric focusing was performed following in-gel re-swelling of immobilized pH gradient strips with 150 mg of 35 S- or unlabelled detergent/urea extracts (Immobiline DryStrip pH 3–10 NL 18 cm (Amersham Pharmacia) or 17 cm ReadyStrip IPG strips pH 3–6 or 4–8 (Biorad)) as described previously (Scadden and Naaby-Hansen, 2001). Second dimension gel electrophoresis was routinely performed in 9– 16% SDS–PAGE gels. The separated proteins were visualized by staining the gels with silver Shevchenko et al. (1996) or the fluorescent dye OGT MP17 (Page et al., 1999). OGT MP17 is an Oxford GlycoSciences (OGS) product with protein staining properties similar to those of the more commonly employed fluorescent dye SyproRuby (Molecular probes). Alternatively, radiolabelled proteins were blotted onto PVDF membranes (Biorad), dried and visualized by phosphoimaging on a Molecular Imager FX scanner (Biorad). The blots were subsequently used for immunoblotting or stained with Coomassie R-250 for triangulation analysis. Spot analysis Gel image analysis and comparisons of the proteins expression levels were performed using the Melanie 3 software (Genebio). Semiquantitation and comparison of radiolabelled protein spot volumes and intensities were performed using a combination of Melanie 3 and the in-house program ChiMap. The values given for each protein spot from different samples were Oncogene

calculated as relative differences compared to the spot values from gels using unstimulated 1542-NP2TX cells as the reference standard. Clustering algorithms were performed using an in-house written programme (ChiClust), which searches for either similarities or differences between every matched protein spots on two gels, and incorporates these differences into a generic % homology value. The ChiClust results are produced in the form of a dendrogram or relational tree as illustrated in Figure 3c. Differentially regulated proteins from 35S-images were aligned with silver- or fluorescence-stained spots using the Melanie 3 software. All computer alignments and spot selections based on densitometry analysis were visually crosschecked by at least two operators. Proteins selected for analysis by mass spectrometry were excised from stained gels by a software-driven robotic cutter (OGS), or by hand. In-gel digestion Tryptic in-gel digestion was essentially performed as described earlier (Benvenuti et al., 2002; Naaby-Hansen et al., 2003). In brief, pieces of 2D gels were destained, dehydrated with 25 mM ammonium bicarbonate (ABC)/50% acetonitrile (ACN), and vacuum-dried. Then, gel pieces were rehydrated with 10 mM DTT/25 mM ABC solution and incubated at 501C for 45 min, followed by the addition of 50 mM iodoacetic acid or iodoacetamide/25 mM ABC solution. The samples were then incubated in the dark at room temperature for 1 h, washed with 25 mM ABC/50% ACN, and vacuum-dried. Samples were subsequently rehydrated and incubated with 30 ng modified trypsin (Promega) in 25 mM ABC overnight at 371C. Peptides were then extracted by adding 50% ACN/5% trifluoroacetic acid (TFA) solution, concentrated by vacuum-drying and resuspended in 5 ml water. Protein identification by mass spectrometry For matrix-assisted laser desorption/ionization (MALDI)mass spectrometry (MS) analysis, the peptide digest mixture was directly spotted on the target plate together with a saturated aqueous 2,5-dihydroxybenzoic acid (DHB) solution. All the samples were analysed on a Reflex III reflector time-offlight (TOF) mass spectrometer (Bruker-Daltonik, Bremen, Germany) equipped with delayed extraction and a Scout 384 ion source. Spectra were obtained by averaging 32–128 singleshot acquisitions. The peak mass lists were submitted to the database search routine MS-Fit to search the nonredundant protein database compiled by the National Center for Biotechnology Information (NCBI), USA. In the cases in which peptide mass mapping by MALDI-MS was unsuccessful or ambiguous, electrospray ionization (ESI)MS/MS analysis was performed. The remainder of the peptide digest mixture was run on a nano-capillary HPLC. After loading the sample, peptide separation was performed on a silica C18 column (LC Packings, Amsterdam, The Netherlands). The HPLC column was coupled to a 50 mm ID fused silica capillary via a zero dead volume connector, which was coupled to the standard nano-LC interface of a Q-Tof hybrid quadrupole orthogonal TOF mass spectrometer (Micromass, Wythenshawe, UK). Mass spectra were acquired using the MassLynx 3.3 software package with automatic precursor ion selection. The fragment ion mass lists were submitted to the Mascot MS/MS ion search routine (Matrix Science, London, UK) to search the nonredundant NCBI protein database. [3H]thymidine incorporation Cells were plated in 96-well plates at a density of 5000 cells/ well and grown overnight in complete medium. Untreated cells


1695 and cells stimulated with IFNs for 72 h were labelled with 1 mCi/ml of [3H]thymidine for 4 h. Following labelling, the cells were washed three times with PBS, and fixed by 20 min treatment with 10% ice-cold trichloroacetic acid (TCA). After three washes with ice-cold 5% TCA, the cells were lysed in a 0.5 N NaOH solution (10 min on ice), followed by subsequent additions of 1 N HCl and 40% TCA (20 min on ice). The resulting solution was filtered through GF/C Whatman paper to trap TCA-insoluble material. The b-emission from the filter paper was measured by scintillation counter. Transfection Green fluorescent protein (GFP), GFP-Nmi fusion protein and Flag-tagged MxA protein were transiently overexpressed in PC3 cells by lipofection, employing lipofectamine (Gibco) according to the manufacturer’s protocol. Following lipofection, the culture medium was changed to RPMI1640 supplemented with either 0.5% FBS or 10% FBS. The cells were grown in this medium or in medium supplemented with 500 U/ ml INFg for the next 24 h, before examination by fluorescence microscopy or immunoblotting. Immunofluorescence Immunofluorescence was performed as described previously Wojciak-Stothard et al. (1999) 1542-NP2TX and 1542-CP3TX were grown on coverslips, stimulated with IFNs as described above, and stained with a-ezrin (BD Transduction laboratory) and rhodamine-phalloidin. Expression of transfected FlagMxA in PC3 cells was detected by staining with a-Flag M2 mouse monoclonal antibody (Sigma) and a-MxA mouse monoclonal antibody. Specimens were visualized with an LSM 510 confocal microscope using a 40 NA 1.3 Neofluar objective (Zeiss, UK) aligned as described previously. Entwistle and Noble (1994). Live transfected cells were stained with 1 mg/ml of Hoechst 33342 for 10 min, and visualized with a Coolview 12 integrating cooled CCD camera (Photonic Science, East Sussex, UK) mounted over an Axiophot microscope fitted with a 63 NA 1.4 oil immersion objective (Zeiss, UK).

[3H]thymidine incorporation. Sustained exposure to INFa for 72 h reduced DNA synthesis by approximately 30% in both cell lines, compared to the synthesis rate in untreated cells (Figure 1a). INFg alone, and simultaneous treatment with INFa and INFg, almost abolished DNA synthesis in 1542-NP2TX cells (o10% of control) (Figure 1a). In contrast, DNA synthesis in 1542-CP3TX cells was only reduced by 60% following INFg treatment and by 80% following exposure to both INFs (Figure 1a). In accordance with the significant growth suppression induced by IFNg, the expression levels of cyclin E were suppressed by IFNg stimulation in both prostate cell lines (Figure 1b). Treatment with INFg also induced a similar increase in the expression of the downstream effector Stat3 in both cell lines (Figure 1b). These findings imply that INFg induces similar receptor activation in the two cell lines. Likewise, no significant discrepancy was observed between the expression levels of p21, p27, p53 and PTEN in the two cell lines by SDS– PAGE immunoblotting of whole-cell extracts (data not shown). 2D gel comparison of protein expression Differentially expressed proteins in the paired normal and cancer cells were identified by computer-assisted densitometry analysis of fluorescence-stained CHAPS/ urea extracts separated on broad pH range, nonlinear IPG/PAGE gels (Figure 2). The expression levels of 65 out of 1367 computer-matched protein spots (4.7%) were twofold or more downregulated in the cancer cell line compared to the normal epithelial cell line, while 75 of the proteins (5.5%) were upregulated by twofold or more in the cancer cells. In all, 13 protein spots with elevated expression and nine protein spots with down-

Immunoblotting Immunostaining of 1D or 2D blots used 1 : 1000 dilutions of aStat3 polyclonal antibody (Santa Cruz), a-p21 (Santa Cruz), ap27 (Cell signalling), a-Erk polyclonal antibody (Santa Cruz), a-phospho-Ser473-Akt antibody (NEB) and a-Akt (NEB), all generated in rabbits. A mouse monoclonal a-hnRNP-K antibody (gift from Dr G Dreyfuss) was employed in 1 : 1000 dilution as was a monoclonal a-p53 antibody (Santa Cruz). Rabbit antiserum to PTEN was used at 1 : 500 dilution, and a 1 : 300 dilution of a-MxA monoclonal mouse antibody, a 1 : 200 dilution of a-Nmi polyclonal rabbit antibody, a 1 : 100 dilution of mouse monoclonal a-ezrin antibody (BD Transduction Laboratory) and 10 mg/ml of mouse monoclonal a-Flag antibody (Sigma) were employed. Immunostained proteins were visualized using ECL (Amersham Pharmacia).

Results Sensitivity to INFs The growth inhibitory effects of either or both INFa and INFg on the paired normal cell line 1542-NP2TX and cancer cell line 1542-CP3TX were monitored by

Figure 1 Effects of INFs on cell proliferation and signalling. (a) 1542-NP2TX and 1542-CP3TX cells were grown in the presence or absence of either or both INFa (500 U/ml) and INFg (500 U/ml) for 72 h, and their DNA synthesis was measured by a [3H]thymidine incorporation assay. DNA synthesis is presented as % changes compared to the synthesis rate in cells grown in the absence of INF. The data represent the average of three independent experiments (range is indicated by bars). (b) Immunoblots of whole-cell extracts from 1542-NP2TX and 1542-CP3TX cells stained with anti-Stat3 and anti-cyclin E antibodies. Although both cell lines displayed a similar expected response to INFg stimulation, the changes were slightly more pronounced in the cancer cells Oncogene


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regulated expression in the cancer cells were identified by MS analysis (Table 1). Interestingly, the expressions of some IFN-inducible proteins including MxA and several forms of tryptophanyl-tRNA synthetase (IFP53) were downregulated in the cancer cells (Table 1 and Figure 2). De novo protein synthesis in the normal and prostate cancer cell lines Biosynthetic labelling with [35S]methionine and -cysteine was employed to identify differential synthesis patterns in the pair of cell lines and to monitor temporal changes induced during INF treatment. Of the 1511 matched protein spots, 176 (11.7%) showed twofold or higher [35S]Met/Cys incorporation in the normal cell line, compared to 140 (9.3%) proteins with a similarly elevated synthesis rate in the cancer cell line (Figure 3b). The number of proteins that could be detected on a single 2D gel autoradiographic image obtained follow-

Figure 2 Two-dimensional gel analysis of protein expression levels in 1542-NP2TX and 1542-CP3TX cells. Representative fluorescence-stained IPG/PAGE gels loaded with 150 mg chaps/urea extracts from cells grown in the presence of 10% FCS. In all, 1367 protein spots were matched by computer comparison of the gel images. The positions and spot numbers of the differentially expressed proteins listed in Table 1 are indicated. Proteins framed by white squares had elevated expression in the 1542-NP2TX cells, while encircled proteins were found to be upregulated in 1542CP3TX cells

Table 1

ing long-term isotope labelling was increased by an average of 20% compared to the number of proteins detectable on a fluorescence-stained gel of the same samples. Notably, the representation of high molecular weight proteins (480 kDa) was significantly improved on 2D autoradiograms (compare Figure 2, 3a and 4). Moreover, only 65% of the protein features detected on autoradiograms could be identified on corresponding fluorescence- or silver-stained gel images by computer comparison analysis. This discrepancy suggests that the B15% newly synthesized proteins that did not correspond (1) acquire new electrophoretic characteristics due to post-translational modifications, (2) are transferred to the insoluble cellular fraction immediately after ER transit, (3) have rapid turnover rates or (4) are present at too low abundance to be detected by fluorescence- or silver-staining procedures. Differential regulation of protein synthesis by INFs INFg treatment induced a time-dependent reduction in the number of newly synthesized proteins in both cell lines (Figure 3b). Protein synthesis rates in the normal cells were more similar to those of the cancer cells than to those in IFNg-stimulated normal cells (Figure 3c), as shown by ChiClust analysis, which compares similarities and differences between every matched protein spot on different gel images. The synthesis patterns induced in the normal cells by exposure to INFg for 36 and 72 h were dissimilar. In contrast, the synthesis patterns induced in the cancer cells treated with INFg for 36 and 72 h clustered in close proximity. However, the treated cancer cells were the most dissimilar patterns by clustering analysis (Figure 3c). These data indicate that INFg-induced protein synthesis is differentially regulated in the two cell lines and that long-term INFg treatment has a more profound effect on protein synthesis in the cancer cells. This observation was supported by the computermediated densitometry analysis. Almost twice as many proteins showed a more than twofold increase or

Identification of proteins differentially expressed in untreated 1542-NP2TX and 1542-CP3TX cells

Proteins with downregulated expression levels in 1542-CPTX cells Protein name HSP70 related protein APG-2 Bip (GRP 78) Trp-tRNA synthetase (IFP53) Trp-tRNA synthetase (IFP53) Trp-tRNA synthetase (IFP53) Trp-tRNA synthetase (IFP53) Leucine aminopeptidase MxA PA28a

Spot ID

NCBI acc. no.

173 397 554 633 743 548 562 434 1373

6226869 121567 32709 135191 11433596 11433596 7705688 4505291 5453990

Proteins with upregulated expression levels in 1542-CPTX cells Protein name HnRNP-K HSPC 223 Fetuin homolog Ezrin Keratin 8 Keratin 8 Keratin 18 Gp 96 Cathepsin D 14.3.3e Major vault protein PSMA5 Calreticulin

Spot ID

NCBI acc. no.

404 1110 349 318 704 561 832 216 1525 1078 108 1818 592

631470 7106836 11276938 87303 87303 106851 11433746 5822085 4507953 4826820 7106387 117501

Differences between the proteins synthesis rates in unstimulated normal and cancer cells are given in Table 2. Similarities and differences between changes in the synthesis rates of 10 selected proteins induced in the two cell lines by long-term INFg treatment are shown in Figure 6 Oncogene


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Figure 4 Electrophoretic migration of proteins with differential regulated synthesis. Representative 2DE autoradiograms of the protein synthesis patterns in 1542-NP2TX (left) and 1542-CP3TX (right) cells grown in the presence of 10% FCS. The positions of the differentially regulated proteins, which were identified by MS analysis, are indicated by white squares (synthesis upregulated in 1542-NP2TX cells) and circles (synthesis upregulated in 1542CPTC cells). The identities of the numbered proteins are given in Table 2

Figure 3 Changes in protein synthesis induced by long-term IFNg treatment of 1542-NP2TX and 1542-CP3TX cells. (a) Representative 2DE autoradiograms of de novo protein synthesis in 1542NP2TX and 1542-CP3TX cells (left), and in cells treated with IFNg for 36 (middle) and 72 h (right), respectively. The cells were exposed to radioisotope-labelled metabolites for the last 18 h of INFg treatment. (b) Comparison of INFg-induced temporal changes in the number of newly synthesized proteins in the cell lines and in the number of protein spots, which displayed a more than twofold increase or decrease in isotope incorporation. Changes are given as a percentage of the total number of protein spots on images obtained following INFg exposure for the noted time period. (c) Clustering analysis of autoradiogram images representing protein synthesis rates in 1542-NP2TX and 1542-CP3TX cells and their alterations by IFNg stimulation. In the resulting relational clustering tree, similar gels are connected by short branches, while increasing subbranching and long branches join gels with diminishing degrees of similarity

decrease in isotope incorporation in response to INFg treatment for 72 h in the cancer cells (Figure 3b). As indicated by the ChiClust analysis, the discrepancy between the protein synthesis rates was less pronounced at the earlier time point (Figures 3b and c). Identification of differentially regulated proteins In all, 31 protein spots, whose synthesis rates were different in the two cell lines or were differentially modulated by INFg treatment, were excised from fluorescence-stained gels, trypsinized and identified by

MS analysis. The proteins and the degree of up- or downregulation in the cancer cells are summarized in Table 2, and their electrophoretic migrations are shown in Figure 4. The majority of the proteins identified belong to one of the following functional groups: proteins involved in chaperone functions, intracellular transport, metabolic pathway regulation, protein degradation and cytoskeletal organization. The synthesis of 23 proteins was elevated in the cancer cells, compared to only four of the 31 listed proteins in the normal cells (Table 2). There was a good correlation between the synthesis rates and the expression levels of most proteins, including hnRNP-K, keratins 8 and 18, major vault protein, calreticulin, 14-3-3e and gp96 in extracts from both cell lines (Tables 1 and 2, and Figures 5–7). However, discrepancies between the synthesis rate and the total expression level of some protein spots were also observed. For example, the total expression levels of cathepsin D and ezrin were significantly higher in the cancer cells than in the normal cells (Table 1 and Figures 5c and 7), despite elevated synthesis rates in the latter cell line (Table 2). Conversely, the expression of proteasome activator subunit 1 (PA28a) was higher in the normal cells, despite its elevated synthesis rate in the cancer cells, and proteasome subunit a type 5 (PSMA5) was more abundant in the cancer cells despite its higher synthesis rate in the normal cells (Tables 1 and 2, and Figures 5 and 6). However, since most post-translational modifications alter the electrophoretic migration of proteins, long-term biosynthetic labelling and fluorescence gel staining experiments must be combined with immunoblotting analysis, before the regulated turnover rate of individual 2D gel protein spots can be interpreted rationally (see Figures 5–7 and below). Temporal changes in protein synthesis induced by long-term exposure of the normal and cancer cell lines to INFg (500 U/ml) are shown in Figure 8. Some of the responses to INFg were similar in the two cell lines. Increased synthesis of keratins 8 and 18 and HSPC223 Oncogene


1698 Table 2 MALDI-MS and ESI-MS/MS identification of proteins with different synthesis rates or differential regulation by INFg in 1542NP2TX and 1542-CP3TX cells Spot ID

Protein name

NCBI gi. no.

108

4826820 MALDI

1.5 times higher

113 216 318

Major vault protein APG-2 gp96 Ezrin like protein

1.2 times lower 1.6 times higher 1.2 times lower

341 349 397 404 421

Transglutaminase Fetuin homolog BiP hnRNP-K ??

434 514

MxA Keratin 6A ISG-54K IFP 53 IFP 53 Keratin 8 Leucine amino peptidase Asparagine synthetase Calreticulin Acyl-CoA hydrolase IFP 53 ERP60 Keratin 8 IFP 53 Keratin 17 Keratin 18 Pyrophosphatase 14-3-3e HSPC223 PA28a Cathepsin D chain B PSMA5 HSP27 Nmi

6226869 ESI 11433746 MALDI 11276938 MALDI and ESI 4759228 MALDI ESI 121567 MALDI 631470 MALDI MALDI and ESI 4505291 MALDI 5031839 ESI 124488 ESI 32709 ESI 32709 MALDI 87303 MALDI 7705688 MALDI

548 554 561 562 571 592 611 633 672 704 743 759 732 1073 1078 1110 1373 1525 1818 1924

Identified by

68656 MALDI 117501 MALDI 325228288 MALDI 32709 2507461 87303 32709 547751 106851 11056044 1168198 7106836 5453990 5822085

Synthesis rate in 1542-CPTX cells compared to 1542 NPTX cells

1.9 times higher nd 2 times higher 1.4 times higher 1.4 times higher 1.7 times higher 1.6 times higher 6.5 2.9 6.6 3.2

times times times times

higher higher higher higher

4.7 times higher 2 times higher nd

MALDI MALDI MALDI MALDI MALDI MALDI MALDI MALDI MALDI ESI MALDI

nd 1.4 times higher 1.4 times higher 4 times higher 1.8 times higher 1.3 times higher 1.6 times higher 1.4 times higher 1.2 times higher 2 times higher 2.2 times lower

7106387 MALDI 19855073 MALDI I.B.

1.2 times lower 1.7 times higher

were observed in both cell lines, and the increases in protein synthesis rates generally were similar in the two cell lines (Figures 8b–d). The synthesis of a third intermediate filament protein, keratin 17, was also elevated in the cancer cells (Table 2) and likewise increased in both cell lines in response to INFg (data not shown). INFg also induced synthesis of PA28a and keratin 6A in both cell lines. While the increased synthesis rates were sustained in the cancer cells during continuous cytokine exposure, their synthesis in the normal cell line declined during the second half of the time course (Figures 8e and f). Other responses to INF were dissimilar between the two cell lines. For example, the synthesis of two different molecular weight forms of tryptophanyl-tRNA synthetase (IFP53) was differentially regulated following INFg treatment. Synthesis of the highest molecular weight form was increased in the normal cells, but decreased in the cancer cells (Figure 8g). Conversely, synthesis of the Oncogene

Figure 5 Differential synthesis rates and expression levels of proteins in 1542-NP2TX and 1542-CP3TX cells. (a) Enlarged area of 2D autoradiograms demonstrating changes in the synthesis of hnRNP-K and keratin 18 induced by either or both INFa and INFg treatment for 72 h. Note the elevated synthesis of both proteins in untreated cancer cells. (b) Enlarged areas of 2D autoradiograms demonstrating differential synthesis of keratin 8 (spot 561 and surrounding related forms, top) and PA28a (bottom) in 1542-NP2TX and 1542-CP3TX cells and their alterations by exposure to INFg for 72 h. Note the significant upregulated synthesis of both proteins in the cancer cells. (c) Comparison of fluorescence-stained 2D gel images demonstrated differential expression levels of keratin 18, hnRNP-K, keratin 8, ezrin, cathepsin D and major vault protein in whole-cell extracts. Note the higher abundance of all six proteins in extracts from the cancer cell line

Figure 6 Discrepancy between synthesis rates and expression levels of MxA in the normal and cancer cells. (a) Metabolic labelling demonstrated increased MxA synthesis in the cancer cell line (the radiolabelled spot corresponds to spot #1 in the 2D immunoblotting images), while immunoblotting demonstrated elevated expression in the normal cell line and that MxA was upregulated in both cell lines following INF interferon treatment (b). (c) INFg treatment induced acidic post-translational modifications of MxA in both cell lines and the appearance of three new isoforms (first dimension electrophoresis performed on pH 4–6 IPG strip). Note the low abundance of MxA in untreated cancer cells


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existence of two alternative forms of the IFP53 gene product, and while the underlying specific post-translational modification of IFP53 is facilitated in one cell line, it is reversed in the other. Both cell lines responded to INFg by similar reductions in the synthesis rate of APG-2, a member of the HSP70 chaperone family (Figure 8i). The synthesis of major vault protein was also reduced in both cell lines by INFg treatment, although at different rates (Figure 8j). Discrepancy between synthesis rates and expression levels of the INF-inducible MxA

Figure 7 Differential expression of hnRNP-K, Nmi, ezrin, gp96, c-Myc and 19S subunit S10B components, demonstrated by immunoblotting. (a) Treatment with INFa, INFg and their combination increased the expression of hnRNP-K in 1542NP2TX, with INFg as the strongest inducer. In contrast, INF treatment did not alter or even slightly suppressed the proteins expression in 1542-CP3TX cells. (b) INFa, INFg and their combination induced Nmi expression in both cell lines, but more than twice as much in the normal cell line. (c) 2D immunoblotting with anti-ezrin antibody revealed the existence of seven ezrin forms. Four ezrin isoforms were detected in unstimulated normal prostate cells, while six isoforms were found in the extracts from untreated cancer cells. INFg induced a seventh acidic isoform in the cancer cells and the weak appearance of two additional isoforms in the normal cell line, which represent the two most acidic forms easily detectable in untreated cancer cells. The ezrin isoforms are evenly spaced, suggesting that they are derived by phosphorylation. (d) Gp96 expression is upregulated in untreated cancer cells. (e) Upregulated expression of c-Myc protein in the cancer cells detected by immnuno stain with monoclonal antibody. (f) Elevated expression of 26S proteasome ATPases (subunit S10B) in the cancer cells demonstrated by 2D immunoblotting. INFg treatment increased the expression of all four 19S subunit ATPases in both cell lines. (g) Differential expression and induction of Nmi in PC3, LNCaP and DU145 cells, demonstrated by SDS–PAGE immunoblotting

lower form was elevated in the cancer cells, but decreased in the normal cells (Figure 8h). Interestingly, an inverse correlation between the average increase and decrease in the synthesis rates of the two forms was observed in each cell line (compare trendlines in Figures 8g and h). One explanation for such distinct but temporally connected regulatory patterns could be the

The synthesis rate of the INF-inducible GTPase MxA was significantly elevated in the cancer cells compared to the normal cells (Table 2 and Figure 6a). In contrast, the expression of MxA was found to be higher in the normal cells. Due to its low abundance in untreated cells, visualization of the MxA spot by gel staining procedures can be difficult (spot 434 in Figure 2). Immunoblotting experiments confirmed the increased abundance of MxA in the normal cells (Figure 6b). MxA was localized to the cytoplasm in both cell lines by immunofluorescence staining, and its localization was unaltered following long-term exposure to IFNg (data not shown). Immunoblotting experiments demonstrated increased MxA expression in both cell lines in response to either INFa or INFg (Figure 6b). A single MxA form was detected in extracts from unstimulated normal and cancer cells. IFNg induced a significant increase in acidic posttranslational modifications of MxA and resulted in the appearance of three new isoforms on immunoblots from narrow pH-range 2D gels (Figure 6c). The two most acidic MxA forms were slightly more pronounced in extracts from INFg-treated cancer cells than in extracts from identically treated normal cells (Figure 6c). The multitude and equal distance between the new isoforms suggest that INFg induces MxA phosphorylation at more than one site. Downregulated expression of Nmi in cancer cells appears to be a consequence of the cells’ elevated PI3K activity N-myc interacting protein (Nmi) was originally identified as a binding partner to the Myc family proteins Max and Mxi (Bao and Zervos, 1996). Nmi lacks an intrinsic transcriptional activation domain, but augments transcription in both IL-2- and IFNg-mediated signalling by acting as an adaptor, which enhances the association between CBP/p300 coactivator proteins and Stats 1 and 5 (Zhu et al., 1999). The expression level of Nmi is downregulated in the cancer cells compared to the normal cells, but is induced in both cell lines by INFs, although to a lesser degree in the cancer cells (Figure 7). Downregulation of the INF-regulated gene activator Nmi may explain the repressed expression of INFinducible genes observed in the cancer cells. Ectopically expressed GFP-tagged Nmi protein was mainly localized in cytoplasm with a punctate, granular pattern as shown previously in 293 and Kelly cells Oncogene


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Figure 8 Changes in protein synthesis induced by sustained INFg treatment of 1542-NP2TX and 1542-CP3TX cells. Cathepsin D chain B serves as a negative control as its synthesis rates in both cell lines were unaffected by INFg treatment

(Bannasch et al., 1999). Interestingly, besides cytoplasmic localization, we also observed nuclear localization of the fusion protein. Overexpression of Nmi led to detachment of the cells from the tissue culture dishes and induced chromosome condensation and/or fragmentation. Although the exact mechanism underlying Nmi-induced detachment still remains unknown, it is tempting to speculate that surplus of Nmi may antagonize its normal physiological role as an adaptor protein, which promotes association between CBP/p300 coactivator proteins and Stats 1 and 5, by disrupting the optimal ratio between the binding partners. Nmi expression was examined in a panel of prostate cancer cell lines and an inverse correlation between Nmi levels and Akt activity was demonstrated, suggesting that Nmi expression is negatively regulated downstream of Akt. The expression level of Nmi was highest in DU145, followed by PC3, and almost undetectable in LNCaP (Figure 7). LNCaP and PC3 cells are known to possess deletions or mutations in PTEN, leading to constitutive activation of Akt (more so in LNCaP than in PC3), while DU145 cell has retained wild-type PTEN and thereby the lowest PI3K activity (Nesterov et al., Oncogene

2001). The activity of the PI3K/Akt pathway was higher in untreated 1542-CP3TX cells than in 1542-NP2TX cells (data not shown), further supporting the existence of an inverse correlation between Nmi expression and the activity of the growth-promoting PI3K/Akt pathway. This finding suggests that Nmi activity may be involved in the implementation of INFg-induced growth inhibition. Both synthesis and expression of hnRNP-K were elevated in cancer cells, but only altered in normal cells by INFg treatment A differentially regulated 57 kDa protein spot with a pI of 5.3. was identified as heterogeneous nuclear ribonucleoprotein K (HnRNP-K), a regulator of transcriptional activity partly controlled by the MAP kinase Erk and the Src kinase family of proto-oncogenes (Habelhah et al., 2001, Ostareck-Lederer et al., 2002). The synthesis rate of HnRNP-K was significantly elevated in the cancer cells (Figures 5a and 8l). In accordance, a threefold higher expression of hnRNP-K was detected in the cancer cells by both immunoblotting and


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fluorescence gel staining of whole-cell extracts (Figures 5c and 7). hnRNP-K expression has previously been shown to be regulated by EGF signalling (Mandal et al., 2001) and, in agreement with this finding, EGF induced higher activity of both MAPK and PI3 K in the prostate cancer cells than in the normal cells (Figure 9). hnRNP-K was differentially regulated by INF in the two cell lines. Both the synthesis rate and expression of hnRNP-K were increased following treatment of the normal cells with either or both INFa and INFg for 72 h (Figures 5a, 7 and 8l). Conversely, neither the synthesis nor expression of hnRNP-K was elevated by long-term INF treatment of the cancer cells (Figures 5a and 7). hnRNP-K binds and is phosphorylated by an IL-1responsive kinase (Van Seuningen et al., 1995), but to our knowledge this is the first study to demonstrate that hnRNP-K synthesis and expression are modulated by INFs. ISGF3g is differentially regulated by INF and EGF ISGF3g, the positive regulator of INFa/b-induced transcription, was inversely regulated by INFa and INFg in the normal and cancer cell lines (Figure 9). ISGF3 was induced by INFa and to a lesser extent by INFg treatment of the normal cell line, and this ratio was not altered when the cells were exposed simultaneously to INF and EGF. However, not only did INFg have a stronger effect on ISGF3 expression in the cancer cells, than did INFa, ISGF3’s induction by INFg was further enhanced by simultaneous stimulation with EGF (Figure 9). These results suggest that the cancer cells JAK/STAT signalling is abnormally influenced by an enhanced activity in EGF-regulated pathways.

Figure 9 (a) Increased phosphorylation of Erk demonstrated by charge-shifts on 2D immunoblots. Note that increased Erk phosphorylation in the cancer cells leads to acidic (left) shifts in the isoforms migration (oblique arrows). Both cell lines responded to INFg with a change in Erk phosphorylation, demonstrating crosstalk between the JAK/STAT and MAPK pathways. (b) Increased activity of the PI3K/Akt pathway in the cancer cells, demonstrated by immunoblotting with an antibody to phosphoAkt. (c) Regulation of ISGF3g expression by INFs, EGF and their combinations, demonstrated by immunoblotting

Discussion Protein expression and synthesis have been compared in a pair of normal and cancer cell lines derived from the same radical prostatectomy specimen. Elevated expression of cathepsin D, c-Myc protein, major vault protein, hnRNP-K, leucine aminopeptidase and ezrin was seen. Approximately one-third of the differentially regulated proteins identified in this study are involved in holding, folding and degradation of cellular proteins. APG-2, gp96, grp78, protein disulfide isomerase (ERP60), calreticulin (Crt), leucine aminopeptidase (LAP), cathepsin D chain B, proteasome activator subunit 1 (PA28a), proteasome 19S subunit S10B and 20S proteasome z subunit (PSMA5) are involved in either chaperone-assisted protein folding in the endoplasmic reticulum (ER), specific functional cleavage of proteins, protein degradation via the 26S proteasome or antigen presentation. Disruption of normal ER function by accumulation of misfolded proteins (due to mutations, stress or viral infections) or unassembled subunits of multimeric protein complexes activates a signal transduction pathway referred to as the ER stress response (ERSR) (Tirasophon et al., 1998; Barbosa-Tessmann et al., 2000). Activation of ERSR induces de novo synthesis of several proteins, including asparagine synthetase (AS) and ER chaperones such as grp78 (BiP), gp94 (gp96), Crt and ERP60 (Tirasophon et al., 1998; BarbosaTessmann et al., 2000). Misfolded proteins are singled out and retained inside the ER, mostly by interactions with chaperones, and eventually targeted for degradation referred to as ER-associated degradation (ERAD) (Kopito, 1997; Frigerio and Lord, 2000). Cytosolic 26S proteasome is the ultimate site for degradation of such defective proteins or orphan protein subunits from the ER (Delom et al., 2001). The synthesis rates of the above-mentioned ERSR-induced proteins were all upregulated in the cancer cell line (Table 2), as were the expression of 19S subunit S10B ATPases (Figure 7). These ATPases, which are situated at the interphase between the 19S regulatory cap and the 20S proteolytic barrel of the 26S proteasome, provide the energy necessary for transport and proteolytic degradation of the proteasomes ubiquitinated substrates. Our findings suggest that ER-associated protein degradation is elevated in the cancer cells, and thus provides a putative explanation for how the observed discrepancy between some proteins synthesis rates and their expression levels in the two cell lines may be established (e.g. MxA; Figure 6). Protein degradation is the source of antigenic peptides presented on the cell surface by MHC class I molecules, and IFNg is known to promote antigen presentation by MHC class I through multiple actions, including the induction of specific proteasome subunits (Rock and Goldberg, 1999). For example, the expression of proteasome 19S subunit S10B components was induced by IFNg in both cell lines (Figure 7). Gp96 acts in a fashion similar to MHC class I molecules, by presenting cell-specific antigens generated Oncogene


1702

by proteasomal degradation (Suto and Srivastava, 1995). It is known that in INF-mediated immunotherapies, many patients fail to respond to INF (Kuratsukuri et al., 2000). Our finding that gp96, originally identified as an elicitor of resistance to tumor transplantation (Maki et al., 1990), is specifically downregulated in the cancer cell line by INFg may thus provide a possible explanation for why INF treatment fails to induce antitumor activity in some patients, and how deregulations may help cancer cells evade immune responses in the host. The multiple deregulations found in the cancer cell line may explain why they are resistant to the growth inhibitory effects of IFNg. Firstly, overexpression of ISGF3g, which is required for the transcription of INFa/b-inducible genes (Nakaya et al., 2001), and suppression of the transcription regulator Nmi, which interacts with Stats and augments INFg-mediated signalling and gene activation, mean that the signal thresholds necessary for activation of INF-inducible genes are altered in these cells. The activity of the JAK/ STAT pathway may also be modulated by crosstalk with other signalling pathways to a greater extent in the cancer cells, as indicated by their altered ISGF3 expression in response to EGF, further changing their transcriptional response to a signal of a given strength. Secondly, the activity of the PI3K/Akt survival and growth pathway was also found to be elevated in the cancer cell line, as were the synthesis and expression of major vault protein (MVP) (Tables 1 and 2, and Figure 5c). MVP was recently shown to bind the tumor suppressor PTEN in a two-hybrid screen, and a significant portion of endogenous PTEN was found to be associated with vault particles in human HeLa cells (Yu et al., 2002). Although PTEN can dephosphorylate both tyrosine and threonine/serine residues, the tumor suppressive functions of PTEN are linked primarily with its lipid phosphatase activity. PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate at the inner leaflet of the plasma membrane, and by downregulating these PI3K-generated anchorage sites for signal complexes it inhibits the cells growth and survival capacity. Sequestration of PTEN away from its main site of action by upregulated vault particles may thus at least partly explain the increased PI3K/Akt activity and growth potential seen in the cancer cells. The inverse correlation between Nmi expression and the activity of the PI3K/Akt pathway may explain how elevated PI3K activity provides resistance to the growth inhibitory effects of INFg in 1542-CP3TX cells. INFg inhibits growth in embryonic fibroblasts by Stat1mediated suppression of c-myc expression (Ramana et al., 2000). The Nmi adaptor protein is known to facilitate association between the Stat1 transcription factor and its nuclear binding partners (Zhu et al., 1999), and it is tempting to speculate that Nmi also is involved in Stat1 homodimers interaction with the c-myc promotor. Thirdly, the multifunctional protein hnRNP-K was strongly overexpressed in the cancer cell line. hnRNP-K interacts with specific nucleic acid sequences (on ssDNA Oncogene

and RNA) via its three KH domains (Tomonaga and Levens, 1995; Braddock et al., 2002), and proline-rich sequences situated between the KH2 and KH3 domains facilitate interaction with SH3 domain containing proteins. hnRNP-K’s multiple binding partners include members of the Src family of proto-oncogenes and transcription regulators such as TATA binding protein (TBP) and various zinc-finger-containing transcriptional repressors (Bomsztyk et al., 1997). hnRNP-K can act both as an activator and a repressor of transcription and as a translational repressor involved in regulated mRNA silencing (Ostareck et al., 1997; Da Silva et al., 2002; Ostareck-Lederer et al., 2002). hnRNP-K activates transcription of the c-myc gene following binding to the specific CT element of its promoter (Nakielny and Dreyfuss, 1999). The elevated expression of hnRNP-K seen in the cancer cells was indeed accompanied by increased abundance of the proto-oncogene c-Myc (Figure 7). Activation of genes downstream the overexpressed C-Myc transactivator leads to cell cycle progression. The expression of hnRNP-K is regulated by the EGF family of growth factors, and hnRNP-K induction by EGF and heregulin-b1 can be blocked by anti-EGF receptor antibody (Mandal et al., 2001), further supporting the notion that the activity of the EGF-regulated signalling pathways is increased in the cancer cell line. hnRNP-K enhances the proliferation rate of MCF7 cells, probably by stimulating transcription from the c-myc promotor (Mandal et al., 2001). Overexpression of the growth-promoting hnRNP-K and c-Myc proteins in the cancer cells may thus contribute to their resistance to growth inhibition. The hnRNPs K and E1 repress the translation of lipoxygenase (LOX) mRNA during the early stages of erythropoiesis by specific interactions with a control element in the 30 untranslated region of the LOX mRNA (Ostareck et al., 1997). This highly specific interaction between mRNA and hnRNPs blocks the ribosome subunits ability to assemble on the mRNA. The elevated expression of hnRNP-K in the cancer cell line may of course be circumstantial, but it is tempting to speculate that increased activity of this known regulator of transcription and translation may be involved in the observed deregulation of INF-inducible genes seen in these cells, perhaps through repression of a key regulator in the INF signalling pathway. In this respect it is interesting that INFg was recently shown to activate a translational inhibitor of ceruloplasmin mRNA in U937 monocytic cells. The unknown repressor bound to a structural element in the mRNA 30 untranslated region (Sampath et al., 2003). Overall, these results suggest that the exclusion of proteins by the ER quality control system, crosstalk between the EGF- and INF-induced signalling pathways and the regulation of INF-inducible genes are all altered in the prostate cancer cells. Acknowledgements We thank Dr Suzanne L Topalian for providing us with the prostate cell lines 1542-NP2TX and 1542-CP3TX, Dr Warren Leonard for the kind provision of a-Nmi rabbit polyclonal


1703 antibody, Dr Manfred Schwab for providing GFP-tagged Nmi cDNA, Dr Gideon Dreyfuss for providing a-hnRNP-K mouse

monoclonal antibody and Dr Otto Haller for providing aMxA antibody and Flag-tagged MxA cDNA.

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