Autoantibody Signature in Human Ductal Pancreatic Adenocarcinoma Barbara Tomaino,†,‡ Paola Cappello,†,‡ Michela Capello,†,‡ Claudia Fredolini,†,‡ Antonio Ponzetto,§ Anna Novarino,| Libero Ciuffreda,| Oscar Bertetto,| Claudio De Angelis,⊥ Enzo Gaia,£ Paola Salacone,£ Michele Milella,¢ Paola Nistico` ,× Massimo Alessio,# Roberto Chiarle,† Maria G. Giuffrida,¥ Mirella Giovarelli,†,‡ and Francesco Novelli*,†,‡ Center for Experimental Research and Medical Studies (CeRMS) and University of Turin, San Giovanni Battista Hospital, Turin, Italy, Department of Medicine and Experimental Oncology, University of Turin, Turin, Italy, Department of Gastro-Hepatology, Department of Gastroenterology, and Centro Oncologico Ematologico Subalpino (COES), San Giovanni Battista Hospital, Turin, Italy, Department of Internal Medicine, Gastroenterology Unit, San Luigi Gonzaga Hospital, Orbassano, Italy, Laboratories of Immunology and Medical Oncology, Regina Elena National Cancer Institute, Rome, Italy, Proteome Biochemistry, San Raffaele Scientific Institute, Milan, Italy, and CNR-Institute of Science of Food Production (ISPA), Bioindustry Park Canavese, Colleretto Giacosa, Italy Received May 14, 2007
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-year survival rate. Biomarkers for its early detection are lacking. This study was designed to use a proteomics-based approach as a means of identifying antigens that elicit a humoral response in PDAC patients. Antibodies against PDAC-associated antigens are useful for early cancer diagnosis and therapy. Proteins from PDAC cell lines were separated by 2-DE, and the serum IgG reactivity of 70 PDAC patients, 40 healthy subjects (HS), 30 non-PDAC tumor patients, and 15 chronic pancreatitis (CP) patients was tested by Western blot analysis. Spots specifically recognized by PDAC sera and revealed by mass spectrometry corresponded to metabolic enzymes or cytoskeletal proteins. Most were upregulated in PDAC tissues. Thus, it seems that metabolic enzymes and cytoskeletal proteins are specific targets of the humoral response during PDAC. The results of further studies of these serological-defined antigens could be of diagnostic and therapeutic significance in PDAC. Keywords: pancreatic ductal adenocarcinoma • autoantibody • tumor associated antigen • mass spectrometry • two-dimensional electrophoresis
Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor with a very low survival rate. Due to its early and asymptomatic invasion and metastasis, 80% of patients are inoperable at diagnosis and die within a few months. Only patients with a small resectable cancer survive 5 years.1 Unfortunately, it is impossible to diagnose PDAC at an early stage due to the lack of a reliable marker. Serum marker CA19.9 * To whom correspondence should be addressed. Prof. Francesco Novelli, Ph.D., Center for Experimental Research and Clinical Studies (CeRMS), San Giovanni Battista Hospital, Via Cherasco 15, 10126 Turin, Italy. Telephone, (+39) 011-6334463; Fax, (+39) 011-6336887; E-mail,
[email protected]. † CeRMS. ‡ Department of Medicine and Experimental Oncology. § COES. | Department of Gastro-Hepatology. ⊥ Department of Gastroenterology. £ Department of Internal Medicine. ¢ Laboratory of Immunology. × Laboratory of Medical Oncology. # Proteome Biochemistry. ¥ ISPA-CNR. 10.1021/pr070281a CCC: $37.00
2007 American Chemical Society
is used for the diagnosis of pancreatic cancer but is poorly sensitive and specific in that its levels are also high in acute and chronic pancreatitis, hepatitis and biliary obstruction. There is thus a need for better early detection systems. The search has been made for biomarkers by analyzing gene overexpression or protein elevation in pancreatic juice or in sera has led to the identification of several tumor targets, including mesothelin, macrophage inhibitory cytokine-1 (MIC1), and osteopontin.2 Identification of overexpressed and/or aberrantly localized proteins that induce an antibody response in cancer patients could be of assistance in the discovery of new tumor-associated antigens (TAA). Autoantibodies to a number of oncogenic proteins in tumor patients have been reported.3-6 Because genetic alterations guide tumor progression, the antibody response arising in a tumor-bearing host may reflect and identify genetic or protein level changes important for cancer progression.7 An antibody-dependent response against tumor proteins could thus be used to detect low antigen concentrations and indicate imminent tumor progression. In addition, antigens able to induce IgG responses Journal of Proteome Research 2007, 6, 4025-4031
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Published on Web 09/08/2007
research articles imply activation of CD4+ T helper cells.8-10 Thus, identification of TAA that induces circulating IgG may render them more suitable for specific immunotherapy.11 In this study, we screened the reactivity of IgG in PDAC sera against proteins from PDAC cell lines (CF-PAC-1, MiaPaCa-2, and BxPC-3) resolved by 2-DE. By 2-DE Western blot (WB) analysis, the reactivity of IgG from PDAC sera was compared to that of IgG from sera of chronic pancreatitis (CP), non-PDAC tumor patients, and healthy subjects (HS). Proteins specifically recognized by sera from PDAC patients were identified by mass spectrometry (MS) and found to be metabolic enzymes and cytoskeletal proteins. Most were up-regulated in PDAC tissues. The selective presence of IgG to PDAC-associated antigens could be of assistance for the development of new diagnostic tools. In addition, our data suggest that these proteins may play a role in PDAC carcinogenesis, thereby providing a set of PDAC associated antigens to be validated for use a immunotherapeutic targets in PDAC.
Experimental Section Sera Specimens. Serum samples were isolated from venous blood with the informed consent of patients and healthy donors. The Ethical Committees of the Dept. of Internal Medicine, University of Turin and Medical Oncology and Laboratories of Immunology, Regina Elena National Cancer Institute, Rome, approved the study protocol. Samples were stored at -80 °C until use. Sera from 70 PDAC patients (31 M, 39 F, aged 32-86; mean ( SD, 67 ( 11) were tested: 17 clinical stage II, 17 stage III, and 36 stage IV according to the classification of the Union International Contre le Cancer (UICC).12 The reactivity of these sera was compared with that of sera from 40 HS (14 M, 26 F, aged 57-87; mean ( SD, 70 ( 7) with no prior history of cancer or autoimmune disease, sera from 30 patients with other non-PDAC cancers (9 with hepatocellular carcinoma, 12 with breast cancer, 8 with colon cancer, and 1 with ovarian cancer; 11 M, 19 F, aged 44-79; mean ( SD, 66 ( 10); sera from 15 CP patients (9 M, 6 F, aged 49-76; mean ( SD, 59 ( 8). There were no significant differences between PDAC, HS, and non-PDAC age and sex distribution as evaluated by an unpaired 2-tailed Student’s t test and by Fisher’s exact test, respectively (PDAC patients vs HS age P > 0.1, sex P > 0.4; PDAC patients vs non-PDAC patients age P > 0.7, sex P > 0.5). Because CP occurred at a younger age (around 44 years) than PDAC, the age (but not the sex) distribution of these two groups was significantly different (PDAC patients vs CP patients age P > 0.009, sex P > 0.4). Sample Preparation. Cells (107) from the CF-PAC-1, (metastatic, ECACC ref no. 91112501), MiaPaCa-2 (undifferentiated ECACC ref no. 85062806), and BxPC-3 (poorly differentiated, ECACC ref no. 93120816) cell lines derived from PDAC patients were harvested and washed with Hank’s balanced salt solution (Sigma, MO). The pellets were freeze-dried overnight and stored at -80 °C. They were resuspended in 200 µL of rehydration buffer, 5 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer nonlinear pH 3-10 (GE Healthcare Bio-Sciences, Uppsala, Sweden), 80 mM DTT, 10 µL/mL inhibitory cocktails, 1 mM PMSF, and 10 µL/mL nuclease mix (GE Healthcare BioSciences). Protein concentration was measured with the Bradford assay (BioRad, Hercules, CA). 2-DE and WB with Sera. Protein lysate was loaded by in gel rehydration onto Immobiline Dry strips (pH 3-10 NL; 7 cm) (GE Healthcare Bio-Sciences) for analytical (100 µL lysate) and 4026
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Tomaino et al.
preparative (150 µL lysate) gels, respectively. Isoelectric focusing (IEF) was performed on an IPGphor IEF unit system (GE Healthcare Bio-Sciences) with a voltage gradient up to 5000 V for a total of 16 000 Vh. Prior to SDS-PAGE, the IPG strips were equilibrated for 15 min with a solution of Tris/HCl buffer (5 mM; pH 8.8), urea (6 M), 30% glycerol, 2% SDS, and 2% DTT, and then for a further 5 min in the same buffer containing 2.5% iodoacetamide and Bromophenol Blue instead of DTT. For the second dimension, strips were run on small NuPAGE Novex 4-12% Bis-Tris Zoom precast gels (Invitrogen, Groningen, The Netherlands) using Novex X-Cell II Mini-cell system (Invitrogen) at a constant 200 V and transferred onto a Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Sciences) using Novex X-Cell II Blot Module (Invitrogen), or silver stained for mass spectrometry according to Shevchenko.13 The isoelectric point (pI) values of the protein spots were estimated from their position on the 2-DE gel with pH gradient graphs provided by GE Healthcare Bio-Sciences. The molecular mass of the proteins was calculated by comparison with the migration of SeeBlue Plus2 Prestained standard (Invitrogen) of known molecular mass. 2-DE gel images were acquired with “ProXPRESS 2D” (Perkin-Elmer Life and Analytical Sciences, Boston, MA), with a 16-bit slow scan CCD camera cooled to -35 °C and recorded in TIFF format. Blotted membranes were incubated for 15 h at 4 °C with blocking buffer consisting of 5% nonfat dry milk in TBS and then incubated for 4 h with serum at 1:100 working dilution in 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBS-T). After three washes with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 working dilution for 90 min at 20 °C. Immunodetection was accomplished by ECL PLUS (Enhanced Chemiluminescence, GE Healthcare Bio-Sciences). The resulting chemifluorescent signals were scanned with “ProXPRESS 2D” (Perkin-Elmer) with an excitation/emission filter setting of 460/80 and 530/30, respectively, and for an exposure time of 12 s. Images were recorded in TIFF format. Each serum was tested three times on three replica blots. The 2-DE WB images were compared to the 2-DE map images using “ProFinder 2D” (Perkin-Elmer) software to identify the probed spots. Protein Identification by MS. For MS analysis, the spots of interest were excised from preparative 2-DE gels and analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and, when necessary, by electron spray ionization (ESI) ion trap mass spectroscopy. Proteins were destained overnight with a solution of 25 mM NH4HCO3 (pH 8.0) and 50% acetonitrile and then digested in gel with trypsin (Promega, Madison, WI) as previously described.14 For MALDI-TOF MS, 0.5 µL of each peptide mixture were applied to a target disk and allowed to air-dry. Subsequently, 0.5 µL of matrix solution (1% w/v R-cyano-4 hydroxycinnamic acid in 30% acetonitrile, 0.1% TFA) were applied to the dried sample and again allowed to dry. Spectra were obtained with a Bruker Reflex III MALDITOF Mass Spectrometer (Bremen, Germany). The MS spectra interpretation of protein digests was done by “peptide mass fingerprinting” (PMF) using MS-Fit software (http://falcon.ludwig.ucl.ac.uk/MS-Fit.html). For MS/MS experiments, the tryptic peptide mixture was desalted and concentrated in ZipTipC18 devices (Millipore, Billerica, MA). After elution with 60% methanol plus 1% formic acid, 4.5 µL of the tryptic peptide mixture was introduced into a gold-coated borosilicate capillary (Proxeon Biosystems, Odense, Denmark) and analyzed in an
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Autoantibodies Define Tumor Antigens in PDAC
LCQ Thermo (ThermoFinnigan, Waltham, CA) ion trap mass spectrometer fitted with a nano ESI source. The capillary voltage was set to 46 V and spray voltage to 1.8 kV. The most stable signals from the full-scan mass spectrum were trapped and fragmented by low-energy collision-induced dissociation (CID), with normalized collision energy ranging from 22 to 24%. For MS/MS data analysis, the Bioworks 3.0 (ThermoFinnigan) software was used with the human FASTA database (http:// www.ebi.ac.uk/fasta33/). Identification of Proteins by 2-DE WB with Specific Antibodies. To validate the MALDI-TOF MS protein identifications of TPIS, K1C10, and COF1, we performed 2-DE WB with specific antibodies. Nitrocellulose membranes blotted from CF-PAC-1 2-DE gels were blocked with 5% nonfat dry milk in TBS and then incubated in TBS-T for 2 h with the following antibodies: mouse monoclonal anti-triosephosphateisomerase 1 (Abnova Corporation, Taipei, Taiwan), mouse monoclonal antiKeratin 10 antibody (Chemicon International, Temecula, CA), and rabbit polyclonal anti-cofilin-1 antibody (Cell Signaling, CELBIO, Milan, Italy). After three washes with TBS-T, the membranes were incubated for 1 h with either HRP-conjugated goat-anti mouse IgG or HRP-conjugated goat anti-rabbit IgG (both Santa Cruz), as appropriate. Immunodetection and image acquisition were done as previously described. WB Analysis of PDAC Sera Reactivity Against Recombinant COF1. Human recombinant GST tagged-Cofilin-1 (1.5 µg, Upstate, Lake Placid, NY) was run on wells of 10-well small NuPAGE Novex 4-12% Bis-Tris, precast gel (Invitrogen), and transferred to a nitrocellulose membrane as previously described. The blotted membranes were cut into single strips and each strip was separately blocked for 2 h at 20 °C with 5% nonfat dry milk in TBS and then incubated, individually, for 1 h at 20 °C with sera from 19 different PDAC patients that contained antibodies against protein spot number 8 (1:100 working dilution in 5% nonfat dry milk in TBS-T). To identify the exact GST-Cofilin-1 position on the blot, one strip was incubated with rabbit polyclonal anti-cofilin-1 antibody (Cell Signaling, 1:1000). After three washes with TBS-T, the membrane strips were incubated for 1 h at 20 °C with a 1:1000 working dilution of HRP-conjugated rabbit anti-human IgG antibody or HRPconjugated goat anti-rabbit IgG (both Santa Cruz), as appropriate. Immunodetection was accomplished as previously described. WB and Immunohistochemical Analysis of Protein Expression in Tumor and Normal Pancreatic Tissues. Normal pancreatic tissues from patients with surgically treated diseases and PDAC tissues from surgically treated patients were obtained frozen from the Regina Elena National Cancer Institute (Rome, Italy) and used for 1-D WB. Formalin fixed tissues from PDAC patients surgically treated (clinical stage II) and paired normal pancreatic tissues adjacent to tumors obtained from Department of Biomedical Sciences and Human Oncology, University of Turin, (Turin, Italy) were used for immunohistochemical analysis. For WB analysis, fresh frozen tissue was homogenized (T18 basic UltraTurrax, IKA, Wilmington, NC) on ice in 400 µL of lysis buffer containing 50 mM TRIS/HCl pH 7.4, 150 mM NaCl, 0.5% NP40, 0.5% Triton X-100, 1 mM DTT, 10 µL/mL inhibitory cocktails, 1 mM PMSF, and 10 µL/mL nuclease mix (GE Healthcare Bio-Sciences). After sonication with an ultrasound sonicator (Hielscher UP200S, 3 × 40 s, amplitude 40%, cycle 0.5, Hielscher Ultrasonics GmbH, Stuttgart, Germany), the mix was centrifuged (13 000 rpm, 30 min,
4 °C) with the protein solution contained in the supernatant. Twenty micrograms of protein extract, measured with the Bradford assay (BioRad), was run on a 10 wells small NuPAGE Novex 4-12% Bis-Tris, precast gel (Invitrogen) and transferred to a nitrocellulose membrane. Immunodetection was accomplished as described above. For immunohistochemical analysis, fresh pancreatic tumor samples and adjacent normal pancreatic tissues from PDAC patients were fixed using 10% formalin solution, and paraffin-embedded tissues were consecutively sectioned. Briefly, the tissue sections were microwaved in citrate buffer pH 6.0 in a microwave oven for antigen retrieval followed by a washing procedure with PBS. Slides were incubated with the primary antibodies and developed in a Ventana ES automated stainer and 3-3′ diaminobenzidine detection kit (Ventana Systems, Tucson, AZ). Stained sections were counterstained with hemeatoxylin. Results were scored by evaluating the intensity of the staining in the tumor cells compared to the adjacent normal pancreatic tissues as: 0 ) tumor cells were negative for the staining, 1+ ) tumor cells were positive but weaker than the adjacent normal pancreatic tissues, 2+ ) tumor cells were positive with similar intensity to the adjacent normal pancreatic tissues, 3+ ) tumor cells were positive and stronger than the adjacent normal pancreatic tissues. For WB analysis, primary and secondary antibodies were used at 1:1000; for immunohistochemical analysis, they were used at 1:100 working dilution. Antibodies used: mouse monoclonal anti-triosephosphateisomerase 1 (Abnova Corporation), anti-keratin 10 (Chemicon International), anti-elongation factor Tu (Abnova Corporation) antibodies; rabbit polyclonal anti-aldehyde dehydrogenase 1 (Chemicon International); anti-glucose-6-phosphate1-dehydrogenase (Bethyl laboratories, Montgomery, TX), anti-isocitrate dehydrogenase (Biogenesis Ltd, Poole, UK), and anti-cofilin-1 (Cell Signaling) antibodies; HRP-conjugated goat-anti-mouse IgG or HRP-conjugated goatanti-rabbit IgG (both Santa Cruz). Statistical Analysis. All statistics were computed with GraphPad Software Inc. (Version 4, San Diego, CA). Age and sex distribution of PDAC patients and controls was analyzed with an unpaired two-tailed Student’s t-test and Fisher’s exact test respectively. Values are presented as means ( SD. The statistical difference between PDAC and HS frequencies of sera reactivity against antigenic proteins was assessed by MantelHaenszel Chi-square. The differential expression of antigenic proteins in PDAC was statistically compared with normal pancreatic tissues with the unpaired 2-tailed Student’s t-test. Values are expressed as means ( SEM. For all tests a 2-sided p < 0.05 (*) and a p < 0.005 (**) value was considered to be significant and highly significant respectively.
Results Sera from PDAC Patients Contain Autoantibodies to Tumor Proteins. The CF-PAC-1 cell line was used as bait to identify the presence of antibodies to PDAC proteins in the described sera. Proteins extracted from this line were analyzed by 2-DE to obtain a representative 2-DE map (Figure 1A). The protein extracts were also subjected to 2-DE and immunoblotted with sera from PDAC (N ) 70), HS (N ) 40), non-PDAC tumor patients (N ) 30), and CP (N ) 15). Eight spots were recognized solely by serum IgG from PDAC patients (Table 1), their positions were labeled on 2-DE map (Figure 1A) and 2-DE WB (Figure 1B,C). To identify those proteins, the 8 spots were excised from preparative gels and analyzed by MALDI-TOFMS. The identity of the 8 spots was found (Table 2). Proteins Journal of Proteome Research • Vol. 6, No. 10, 2007 4027
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Tomaino et al. Table 1. Frequencies of Sera Reactivity Against Protein Spots in Analyzed Groups number of positive sera spot numbera
PDAC (N ) 70)
non-PDAC (N ) 30)
CP (N ) 15)
HS (N ) 40)
1
10 (14%)b χ2 ) 5.409c, p ) 0.02 16 (23%) χ2 ) 8.457, p ) 0.004 15 (21%) χ2 ) 7.957, p ) 0.005 14 (20%) χ2 ) 7.455, p ) 0.006 9 (13%) χ2 ) 4.888, p ) 0.03 8 (11%) χ2 ) 4.364, p ) 0.04 9 (13%) χ2 ) 4.888, p ) 0.03 19 (27%) χ2 ) 9.937, p ) 0.002
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
2 3 4 5 6 7 8
a Reactive protein spots numbered as showed in 2-DE gel, Figure 1. Percentage of positive sera. c Mantel-Haenszel Chi-square statistical analysis between PDAC and HS, a p-values < 0.05 was considered statistically significant. b
Figure 1. 2-DE map and Western blot of CF-PAC-1 and MiaPaCa-2 cell lines. The proteins from CF-PAC-1 cell line were separated by 2-DE and silver stained (A) or transferred to a nitrocellulose membrane and probed with PDAC patient (B and C) or HS (D) sera. The proteins from MiaPaCa-2 cell line were separated by 2-DE and silver stained (E) or transferred to a nitrocellulose membrane and probed with PDAC patient sera (F, G, and H). The numbered circles indicate the spots specifically recognized by PDAC sera. Their names were listed in Table 2.
identified were members of functional groups consisting of metabolic enzymes: triosephosphateisomerase 1 (TPIS), retinal dehydrogenase 1 (AL1A1), glucose-6-phosphate 1-dehydrogenase (G6PD), elongation Factor Tu (EFTU), and isocitrate dehydrogenase (IDHC) and cytoskeletal proteins: keratin 10 (K1C10), cofilin-1 (COF1) and transgelin (TAGL) (Table 2). Protein identification of COF1 and AL1A1 was confirmed by MS/MS analysis (Table 2). MALDI-TOF analysis revealed the coexistence in spot 8 of TAGL and COF1 (Table 2). The reactivity of the antibodies to COF1 was confirmed by WB with recombinant protein and further verified by WB with a specific antibody (Figure 2A and B, upper panel). Due to the lack of commercially available recombinant TAGL, the reactivity of antibodies to TAGL was not assessed. Further 2-DE WB analysis with specific antibodies confirmed that spots 1 and 2 corresponded to TPIS (further named TPIS1 and TPIS2) (Figure 2B, middle panel) and spot 3 corresponded to K1C10 (Figure 2B, lower panel). 4028
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Figure 2. Validation of PDAC serum reactivity against COF1, TPIS, and K1C10 by WB analysis. The reactivity against COF1 by PDAC sera (N ) 19) was tested on recombinant GST-COF1 by 1-D WB. (A) Representative result of 1-D WB with anti-cofilin-1 antibody, line 1, and with a PDAC serum, line 2. (B) Cropped images from CF-PAC-1 2-DE and 2-DE WB with anti-TPIS, anti-K1C10, and antiCOF1 antibody. The protein spot positions are shown in circles.
To confirm that autoantibodies from PDAC patients recognize proteins that are commonly expressed in pancreatic carcinoma cells at different stages of differentiation, the reactivity of sera for the identified antigen on MiaPaCa-2 and BxPC-3 pancreatic tumor cells was also evaluated. Sera from PDAC patients showed a similar reactivity on these cell lines. By contrast, sera from HS did not show any reactivity to the identified proteins (Figure 1E-H, and data not shown). Expression of Identified Proteins in Tumor and Normal Pancreatic Tissues. To establish whether the identified proteins play a role in pancreatic carcinogenesis, their expression in normal and pancreatic tumor tissues was compared. WB analysis of pancreatic tissues revealed that the expression of TPIS, K1C10, G6PD, EFTU, IDHC, and COF1 was significantly enhanced in PDAC compared to unrelated normal pancreatic (NP) tissues (Figure 3A). By contrast, the already high expression of AL1A1 in normal pancreas was not enhanced in PDAC tissues (Figure 3A). Immunohistochemical analysis confirmed the results for K1C10, AL1A1, G6PD, EFTU, and IDHC (Figure
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Autoantibodies Define Tumor Antigens in PDAC Table 2. Identification of Protein Recognized by PDAC Patient Sera by MALDI-TOF MS pI
spot no.
accession no.a
protein name
1 2 3 4 5 6 7 8
P60174 P60174 P13645 P00352 P11413 P49411 O75874 P37802 P23528
Triosephosphate isomerase (TPIS) Triosephosphate isomerase (TPIS) Keratin, type I cytoskeletal 10 (K1C10) Retinal dehydrogenase 1 (AL1A1) Glucose-6-phosphate 1-dehydrogenase (G6PD) Elongation factor Tu (EFTU) Isocitrate dehydrogenase [nadp] (IDHC) Transgelin (TAGL) Cofilin-1 (COF1)
a
6.4 6.4 5.1 6.3 6.4 7.3 6.5 8.4 8.2
MW (kDa)
matching peptides
coverage (%)
MS FIT score
MS/MSb
26.64 26.64 59.52 54.84 59.26 49.54 46.66 22.39 18.50
12/85 10/62 21/73 12/93 26/64 17/69 15/38 11/58 7/58
53 42 35 35 54 48 40 57 48
9810 2273 5.3 × 105 9372 8.3 × 108 7.5 × 104 5.3 × 105 1.1 × 104 229
1 2
Accession number according to Swiss-Prot. b Number of peptides confirmed by MS/MS.
3B). COF1 and TPIS were not examined by immunhistochemistry because commercially available specific antibodies did not work in formalin-fixed, paraffin-embedded tissues. Immunohistochemical analysis of K1C10, G6PD, and IDHC expression in PDAC tissues showed a strong positive cytoplasmic staining (score 3+) of acinar and ductal epithelial cells compared to NP tissue. By contrast, immunohistochemical analysis of AL1A1 and EFTU expression in PDAC tissues showed a weakly (score 1+) and moderate (score 2+) positive staining, respectively.
Discussion We used a serological approach that combined 2-DE expression profiling of three human pancreatic tumor cell lines (CFPAC-1, MiaPaCa-2, and BxPC-3) and WB with PDAC patient sera to look for autoantibodies to PDAC-associated antigens. We found that from 11 to 27% of PDAC patient sera contain IgG against two functional kinds of proteins: metabolic enzymes (TPIS, AL1A1, G6PD, IDCH, and EFTU) and cytoskeletal proteins (K1C10 and COF1). The number of serologicaldefined antigens identified by our approach is lower than that identified by differential proteome-based analysis. Differential proteome expression profiles of PDAC identified from 11 to 80 up-regulated proteins.15,16 Only eight of them were identified by our serological approach. Similar to our studies, the number of antigens recognized using serological proteome analysis in different types of tumors is below 20% of the up-regulated proteins.17-19 This implies that few of the many differentially expressed proteins up-regulated in these tumors induce a specific and measurable secretion of antibody in vivo. Most of the proteins we identify by the serological approach appear as single spots on the 2-DE map, and do not display posttranslational modifications, with the exception of two TPIS isoforms. This is not a limitation of the serological proteomics method because post-translationally modified proteins have been identified in other serologically based studies.18,20-23 In fact, our methods identified 4 HSP60 isoforms (data not shown), but they were not included in this report because there were no statistically significant differences between their presence in PDAC patient and HS sera. Most of the antigens we identified have been found in a number of proteome-based analyses of distinct tumor types. The expression of the proteins we identified has been reported to be up-regulated in a variety of other tumor tipes.24 For example, the expression of TPIS, COF1, EFTU, TAGL, and IDHC was found to be up-regulated in pancreatic tumors,15,16 and that of AL1A1 and IDHC in hepatic tumors.24,25 K1C10, COF1, AL1A1, and EFTU were found to be up-regulated in renal tumors,24,26,27 and K1C10, EFTU, and IDHC in lung tumors,19 and EFTU and TAGL in stomach tumors.28,29 Last, the expression of EFTU, TAGL, and IDHC were
found to be up-regulated in oesophageal30 tumors and K1C10 in colon tumors.31 These profiles of overexpressed proteins suggest that the aberrant expression of these metabolic and cytoskeletal proteins, is a common feature associated with cancer formation or progression. In our study, antibodies to the identified proteins were not detected in sera from HS and non-PDAC tumor patients, suggesting that the antibody response to these proteins is characteristic of PDAC. However, antibodies to TPIS and K1C10 have been found in sera from lung and kidney cancer patients in other studies.17,19,32 Importantly, in our study, IgG antibodies directed to the identified proteins were not found in sera of patients with CP, indicating that the antibody response to the identified proteins is not related to a pancreatic inflammatory condition, but is specific for neoplasia. The specificity of the antibody response in pancreatic cancer is further revealed by the observation that, with the exception of AL1A1, all the proteins recognized by antibodies in PDAC sera were up-regulated in PDAC biopsies. Tumor proliferation is characterized by an enhanced hypoxic environment and metabolic alterations that favor the increased expression of glycolytic enzymes. In tumor cells, the transcription and translation of G6PD is induced by the key transcription factor hypoxia-inducible factor 1R (HIF-1R). Concomitantly, the enhanced glycolytic activity of tumor cells up-regulates the expression and activity of enzymes such as TPIS,27,33 which in turn favors high levels of pyruvate and lactate production that is the hallmark of the Warburg effect.34,35 Consistent with this, overexpression of glyceraldehyde-3-phosphate dehydrogenase and R-enolase have also been observed in PDAC.36,37 Overexpression of metabolic enzymes in PDAC may allow for enhanced processing and presentation to T lymphocytes by professional antigen presenting cells, thereby overcoming immune tolerance to these self-proteins.38 An interesting point that emerges from our data is that PDAC is characterized by an antibody response to the cytoskeletal proteins K1C10 and COF1. The most frequent antibody response of PDAC patients is directed to COF1, an actin depolymerizing protein involved in invadopodium formation and required for tumor cell directionality in response to chemotactic or growth-factor stimulation.39,40 K1C10 is also involved in formation of the intermediate filament cytoskeleton of all epithelial cells.41 Antibodies to cytoskeletal proteins may reflect the immune system’s attempt to target molecules, such as COF1, involved in tumor metastasis. Although autoantibodies to cytoskeletal proteins have been found in cancer,19,42 autoantibodies to COF1 in PDAC have not been previously described. In this respect, it would be interesting to find out whether the antibodies to cytoskeletal proteins found in PDAC patients inhibit metastatization or tumor cell proliferation. COF1 might Journal of Proteome Research • Vol. 6, No. 10, 2007 4029
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References
Figure 3. Expression of identified proteins in normal and PDAC tissues. (A) 1-D WB analysis of the expression of antigenic proteins in PDAC (N ) 3) compared with NP (N ) 3). Intensity of reactive lines was quantified and expressed as arbitrary units of normalized signal intensity. The results are presented as means of three experiments ( SEM. The significance of the normalized volume intensity of the selected proteins was assessed with the unpaired 2-tailed Student’s t-test. *p < 0.05 vs NP. **p < 0.005 vs NP. (B) Immunohistochemical analysis of K1C10, AL1A1, G6PD, EFTU, and IDHC in representative sections of PDAC and NP tissue adjacent to tumors. Arbitrary score as: negative -, weakly positive 1+, moderatly positive 2+, or strongly positive reaction 3+. (Original magnification 60×).
be functionally associated with TPIS to feed glycolic fuel to Na,K ATPase.43 As the most frequent antibody response in PDAC patients is directed to COF1 and TPIS2, our data support the hypothesis that the two proteins play a role in the biology of pancreatic cancer. The antibody response to serological-defined antigens might have diagnostic value in PDAC. Development of protein arrays able to screen the autoantibody response to the identified proteins might be used to assist in the diagnosis of pancreatic cancer. Further immunological validation studies will establish which of these proteins could be suitable targets for PDAC immunotherapy.
Acknowledgment. This work was supported in part by Compagnia di San Paolo (Special Project Oncology), Ministero della Salute (Programma Integrato Oncologia), Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR), ex-40% Fondo per l’innovazione nella ricerca di base (FIRB), Progetti di Ricerca Finalizzata Regione Piemonte and RiboVax Biotechnologies (Geneva, Switzerland). B.T.’s PhD student fellowship was supported from Bioline srl. P.C. was supported by a fellowship from FIRC. We thank Dr. John Iliffe and Dr. Marianne Murphy for critically reading the manuscript. 4030
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