Serological Immunoreactivity against Colon Cancer Proteome Varies

ohio1/ypr-ypr/ypr-ypr/ypr00208/ypr2551d07z xppws 23:ver.3 1/17/08 11:50 Msc: pr-2007-00360m TEID: nlr00 BATID: pr2a19

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Serological Immunoreactivity against Colon Cancer Proteome Varies upon Disease Progression

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Lucia De Monte,†,‡,§ Francesca Sanvito,| Stefano Olivieri,† Fiammetta Viganò,† Claudio Doglioni,| Matteo Frasson,⊥ Marco Braga,⊥ Angela Bachi,# Paolo Dellabona,§,¶ Maria Pia Protti,‡,§ and Massimo Alessio*,†,§

6 7 8

Proteome Biochemistry, Tumor Immunology, Mass Spectrometry, Pathology, Surgery, Experimental Immunology, Cancer Immunotherapy and Gene Therapy Program, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy

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Received June 11, 2007; Accepted November 7, 2007

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14

Sera from colon carcinoma patients were used to identify tumor-associated antigens (TAAs) by screening tumor proteome resolved by 2D electrophoresis. A panel of six TAAs eliciting a serological immune response in colorectal cancer was identified, showing a modification in antigen recognition by B cells in patients as a function of colon cancer progression. The expression of these proteins was either confined or increased in tumor as compared to normal mucosa.

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Keywords: 2D electrophoresis • colorectal carcinoma • proteomics • tumor-associated antigens

3 4

11 12 13

16

Introduction

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The immune recognition of self-proteins in cancer reflects the attempts of the host immune system to eliminate cells expressing qualitatively and/or quantitatively aberrant proteins. These proteins are defined as tumor-associated antigens (TAAs), which can be recognized by both T and B cells of the immune system of the host. The identification of TAAs is indispensable for the characterization of spontaneous antitumor responses, as well as for the design of immunotherapeutic approaches aiming at supporting the spontaneous responses. Furthermore, TAAs expressed at different stages of the disease can become useful biomarkers for prognosis or diagnosis of the cancer. The humoral immune response to cancer has been documented in humans by the identification of autoantibodies directed against different intracellular and surface antigens in patients affected by various tumor types.1 The use of patients sera allows for the screening of large expression systems based on tumor-derived cDNA libraries (SEREX) or proteome (SERPA). Using these strategies, it is possible to identify many target antigens in one time and have a comprehensive view of the B cell response against the autologous tumor in each patient at any given stage of the disease. Nevertheless, the role of antibodies specific to tumor-related antigens in antitumor immunity still needs investigation. It is also not completely clear whether the antibody response to

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* To whom correspondence should be addressed: Proteome Biochemistry, DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. Telephone: 39-02-26434725. Fax: 39-02-26434153. E-mail: [email protected]. † Proteome Biochemistry. ‡ Tumor Immunology. § Cancer Immunotherapy and Gene Therapy Program. | Pathology. ⊥ Surgery. # Mass Spectrometry. ¶ Experimental Immunology.

504 Journal of Proteome Research 2008, 7, 504–514 Published on Web 01/08/2008

TAAs changes upon progression of the disease, revealing the possible modulation of B cell responses as a function of tumor progression. Furthermore, it is still unclear whether the spontaneous antibody response elicited by a specific tumor is similar or distinct from that induced by tumors of different origin. Colorectal cancer is the third most common malignancy diagnosed worldwide. Conventional treatments (surgery, chemotherapy, and radiotherapy) for colorectal cancer have improved in recent years, yet individuals with advanced disease still have a poor prognosis.2 Colon cancer is still poorly characterized for its antigen content;3,4 therefore, we have focused on the identification of tumor-related antigens expressed by this type of tumor. To identify proteins eliciting humoral responses in colon cancer patients, a proteome-based approach [serological proteome analysis (SERPA), PROTEOMEX, or serological and proteomic evaluation of antibody responses (SPEAR)] has been applied, allowing for the identification of autoantibodies to proteins as they occur in tumors and tumor cell lines.5–7 The identification of tumor-related antigens uses the reactivity of sera from patients to screen tumor proteome resolved by twodimensional electrophoresis (2DE).5–7 This approach allows for the screening of a large number of sera and the determination of the occurrence of the relevant autoantigens. In addition, SERPA permits the detection of autoantibodies directed against protein isoforms and post-translational modifications. The SERPA-based approach has been successfully applied to different types of cancer, such as renal cell carcinoma,5,8,9 neuroblastoma,10 acute leukemia,11 hepatocellular carcinoma,12 lung,13,14 breast,15 pancreatic,16 and also recently in colorectal cancers.4 Several of the antigenic proteins identified by immunoproteomics have already provided novel cancer biomarker candidates that may be of clinical use concerning diagnosis and prognosis, and large clinical studies are planned to define their value.17,18 These proteins may also constitute 10.1021/pr070360m CCC: $40.75

 2008 American Chemical Society

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Serological Immunoreactivity against Colon Cancer Proteome

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vaccine candidate antigens for immunotherapeutic approaches,9,19 as in the case of renal cell carcinoma, in which tumorrelated antigens identified also by immunoproteomics are included in clinical phase I and II trials.20 Here, we report the identification of a spontaneous humoral response in colon cancer patients compared to control subjects elicited by six proteins, found specifically expressed or overexpressed in tumor cells. Moreover, we describe a modification of the antigenic pattern recognized by antibodies as a function of colon cancer progression.

87

Experimental Section

88

Sera and Tissue Sample Collection and Preparation. Sera samples, obtained after informed patient consent and approval by the Local Research Ethical Committee, were collected at the time of surgical resection from 52 patients with colorectal cancer (Cr: age, 67 SD 11.05; sex, M ) 27, F ) 25) and 9 patients with colon adenoma (Ad: age, 66 SD 9.76; sex, M ) 8, F ) 1) as inferred later by histopathology diagnosis. Control sera were obtained from 40 age- and gender-matched subjects (Cn: age, 65 SD 15.30; sex, M ) 15, F ) 25), including healthy donors (n ) 17) and patients with nontumoral pathologies (n ) 23) (characteristics of patients and controls are described in Tables 1 and 2). Age and gender distribution between patients and controls were not statistically different as evaluated by Student’s t test and Fischer’s exact test. Colon carcinoma patients with or without metastasis in the regional lymph nodes were considered as separated subgroups: colon-carcinoma characterized by metastasis in regional lymph nodes or distant organs, stages III and IV of tumor nodes metastasis (TNM) classification21 (Cr-Stg.III-IV; n ) 27; age, 66 SD 10.83; sex, M ) 13, F ) 14); colon carcinoma with no lymph node metastasis, stages I and II of TNM classification (Cr-Stg.I-II; n ) 25; age, 68 SD 11.28; sex, M ) 14, F ) 11). These subgroups too did not show statistically different age and gender from controls. The adenoma patients, because of the limited number, showed a statistical difference in gender but not in age (Ad versus Cn: age p ) ns, gender p ) 0.008). Sera from 23 pancreatic adenocarcinoma and 9 lung carcinoma patients, including different stages of disease progression, were also used. Samples from cancer and normal mucosa surrounding the tumor were obtained from the resected colon and either paraffin or OCT embedded; the latter were stored at –80 °C. Cell Lines and Cell Culture. Tumor cell lines used in this study were colon adenocarcinoma LS180, HT-29, and LoVo [American Type Culture Collection (ATCC), Rockville, MD]; nonsmall cell lung carcinoma Calu-1 (ATCC), Med, and Dem (established from primary tumor, made available by Dr. Nisticò, Regina Elena Institute, Rome, Italy); renal cell carcinoma Car and Pre (established from a primary tumor in our laboratory); breast adenocarcinoma MCF7, MDA-MB-231 (ATCC), and DAL22 (made available by Dr. Nisticò); and pancreatic ductal adenocarcinoma CFPAC-1 (ATCC), PaCa44 (established by H. Elsasser, provided by Dr. Piemonti, HSR23), and A8184 (Interlab Cell Line Collection, CBA, Genoa, Italy). All cell lines were grown in RPMI containing 10% fetal bovine serum, penicillin, and streptomycin under standard conditions. Two-Dimensional Electrophoresis and Western Blotting. Cultures of tumor cells were collected and solubilized in lysis buffer [8 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail]. Samples (250 µg each) were applied to 7 cm immobilized pH gradient (IPG) strips pH3-10NL (GE-Healthcare, Milan, Italy),

77 78 79 80 81 82 83 84 85

89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138

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and 2DE was performed as reported.24 Second-dimension separation was performed on 10% acrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run on a Mini-Protean 3 Dodeca Cell apparatus (BioRad, Hercules, CA). Proteins were electrotransferred to nitrocellulose membrane and then incubated 12 h at 4 °C with sera obtained from either patients or healthy individuals (1:300 dilution). Immunodetection was revealed with antihuman IgG antibodies conjugated with horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) followed by ECL reaction (GEHealthcare) and autoradiography. Films obtained at 30 s exposure time were used for densitometric analysis. Protein Visualization and Image Analysis. Preparative minigels (300 µg) were stained with colloidal Coomassie Blue (Sigma). Stained gels or autoradiography films were scanned using a Personal SI Laser Densitometer (GE-Healthcare), and 2D protein patterns were analyzed using Progenesis Workstation V2004 software (Nonlinear Dynamics, Newcastle, U.K.). Relative mass (Mr) was estimated by a comparison to reference markers (Precision, BioRad), and pI values were assigned to detected spots by calibration, as described in the GE-Healthcare guidelines. Statistical Analysis. Each serum was tested twice on a 2DE map, and the corresponding images were analyzed. Sera pattern reactivity was compared in patients and controls. Analysis was performed also considering the two groups of patients with or without regional lymph node metastasis. To have identical criteria along the analysis and to avoid subjective evaluation, sera reactivity was considered positive or negative for a single protein spot when spots were detected by Progenesis software under automatic default conditions. Statistical analysis of categorical data (gender distribution and the presence or absence of immunoreactive spots) was performed by 2 × 2 contingency table analysis using Fisher’s exact test and two-tailed p values. Age distribution was evaluated using unpaired Student’s t test with two-tailed p values for the comparison of two means with standard deviation. In all analysis, p < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 4, version 4.03 software (GraphPad, Inc., SanDiego, CA). The sensitivity and specificity of TAA markers were calculated with the receiver-operating characteristic curve (ROC) methodology, as previously described,25 using the web-based calculator for ROC (available at Johns Hopkins University, www.jrocfit.org). Protein Identification. MS analysis was performed as previously described24,26 using a matrix-assisted laser desorption ionization-time of flight (MALDI–TOF) Voyager-DE STR (Applied Biosystem, Foster City, CA). Proteins were unambiguously identified by searching a comprehensive nonredundant protein database National Center for Biotechnology Information (NCBI) and the mass spectrometry protein sequence database (MSDB) selected by default by the software programs used ProFound version 4.10.5 and Mascot in-house version 1.9.00, respectively.27,28 One missed cleavage per peptide was allowed, and an initial mass tolerance of 50 ppm was used in all searches. Protein identity was confirmed by 2D Western blotting using LS180 cell lysates and commercially available antibodies: mouse mAb antivalosin-containing protein (VCP) (Progen Biotechnik, Heidelberg, Germany); rabbit polyclonal anti-R-actinin 4 (Alexis Biochemicals, Lausen, Switzerland); rat mAb anti-Grp94 (Stressgen Biotechnologies, Victoria, British Columbia, Canada); goat polyclonal anti-aldolase A, mouse mAb anti-lamin A/C, and Journal of Proteome Research • Vol. 7, No. 2, 2008 505

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De Monte et al. a

Table 1. Clinical Features of Colorectal Cancer and Adenoma Patients

immunoreactivity detected on 2DE patient

gender

age

stage (category)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9

F M M M M F F F F M M M F F M M M M F F F M F M M F M M F F M F M F M F M F M F M F F M M F F M F F M M M M M F M M M M M

57 53 73 57 71 61 51 53 63 74 90 73 73 60 61 81 84 80 62 57 75 59 70 82 83 61 67 64 52 68 45 74 79 44 80 55 81 83 66 69 73 48 77 66 76 73 60 74 65 66 66 57 56 75 74 70 52 70 78 61 55

stg I (T1N0M0) stg I (T1N0M0) stg I (T2N0M0) stg I (T2N0M0) stg I (T2N0M0) stg I (T2N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3NXM0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T3N0M0) stg II (T4N0M0) stg II (T4N0M0) stg II (T4N0M0) stg I (T2N0M0) stg III (T3N1M0) stg III (T3N1M0) stg III (T3N2M0) stg III (T4N1M0) stg III (T3N1M0) stg III (T4N2M0) stg III (T3N1M0) stg III (T3N1M0) stg III (T3N2M0) stg III (T3N1M0) stg III (T3N1M0) stg III (T3N2M0) stg III (T2N2M0) stg III (T3N1M0) stg III (T3N1M0) stg III (T3N1M0) stg III (T4N1M0) stg III (T4N2M0) stg III (T3N2M0) stg III (T4N2M0) stg III (T3N2M0) stg IV (T4N1M1) stg IV (T4N2M1) stg IV (T4N2M1) stg IV (metas)c stg IV (metas) stg IV (metas) adenoma adenoma adenoma adenoma adenoma adenoma adenoma adenoma adenoma

GRP94

Act4

+ + +

+

VCP

LamC

AldA

+ + +

+

hnRNP AB

+ + + + +

+ + + +

+ + +

+ +

+

+ + + +

+

+ + + + +

+ +

+ +

+ + + + +

+

+

+ + + + +

+

+

+

+

+ +

+ + +

+

+

+ + +

+

+ +

+

+

+ +

+ +

+

+ +

+ + +

+ +

+

+ + +

+

+ +

+ + + +

+

+

a List of colorectal cancer and adenoma patients features with the corresponding 2DE serum reactivity against the identified cancer-related proteins assessed on the LS180 proteome. +, positive reactivity; M, male; F, female. GRP94, glucose-regulated protein 94; Act4, R actinin 4; VCP, valosin-containing protein; LamC, lamin C; AldA, aldolase A; hnRNP AB, heterogeneous nuclear ribonucleoprotein AB. b Staging and category of colon carcinoma according to TNM classifications.21 UICC, tumor, nodes, and metastases: T1-T4, invasive tumors (T1, submucosa; T2, tunica muscularis; T3, subserosa; and T4, peritoneum or other organs). N0, no malignant regional lymphnodes; N1, 1–3 regional lymphnodes metastases; N2, >4 regional lymphnode metastases. M0, no distant metastases; M1, distant metastasis. Stage I (T1–2, N0, M0), stage II (T3–4, N0, M0), stage III (T1–4, N1–2, M0), and stage IV (any T, any N, M1). c Liver metastasis of colon carcinoma.

201 202 203 204 205

rabbit polyclonal anti-hnRNP AB (Santa Cruz Biotechnology, Santa Cruz, CA). Protein Expression in Primary Tumors and Cell Lines. Tumor tissue samples and normal mucosa surrounding the tumor specimens were sliced (15 µm); 15 sections were 506

Journal of Proteome Research • Vol. 7, No. 2, 2008

collected and solubilized with 200 µL of Laemmli buffer in the presence of a protease inhibitor cocktail. Proteins (30 µg/lane) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis (see above). Reactivity with anti-β-actin mAb (Sigma, Milano,

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Serological Immunoreactivity against Colon Cancer Proteome a

Table 2. Features of Control Subjects

immunoreactivity detected on 2DE subject

gender

age

clinical status

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

F F M F M M F F F F M F M M F M M F F F M F F F M M F F F F M F M F F M M F F F

76 79 77 70 44 45 43 57 58 61 73 68 69 71 73 86 73 61 76 59 91 73 67 73 60 81 50 67 62 58 81 62 79 64 72 78 69 30 26 27

rectocele inguinal hernia inguinal hernia diverticulosis healthy healthy healthy thyroid goiter healthy diverticulosis inguinal hernia hepatic cyst rectal prolapse cataract cataract diabetes healthy cholecystitis diabetes endometrial polyp hemorroiditis diabetes healthy healthy healthy pulmonary embolism cervical ectropion healthy atrial fibrillation healthy healthy healthy healthy bronchial asthma radicolitis prostatic hyperthrophia healthy healthy healthy healthy

GRP94

Act4

VCP

LamC

AldA

hnRNP AB

+

+ + +

+ + +

+

+

+

+

+

+

+

a List of control subjects features and 2DE serum reactivity obtained on LS180 cell line. +, positive reactivity; M, male; F, female. GRP94, glucose-regulated protein 94; Act4, R actinin 4; VCP, valosin-containing protein; LamC, lamin C; AldA, aldolase A; hnRNP AB, heterogeneous nuclear ribonucleoprotein AB.

211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229

Italy) was used as a loading control and expression level normalization. The expression level of the relevant antigens in cancer cell lines was assessed on the proteome resolved by 2DE by Western blot performed using specific antibodies (see above). Expression levels were quantified by densitometric analysis; high and low were referred to LS180 expression levels taken as the reference for high intensity. Immunohistochemistry. Formalin-fixed, paraffin-embedded sections from colon carcinoma samples of selected patients and their formalin-fixed, paraffin-embedded normal surrounding mucosa specimens underwent antigen retrieval and quenching with 3% hydrogen peroxide. Sections were incubated 12 h at 4 °C with anti-VCP (1:15 000), anti-aldolase-A (1:500), anti-Grp94 (1:500), and anti-actinin 4 (1:1500). Rabbit anti-goat IgG and anti-rat Ig (Vector Laboratories, Burlingame, CA), followed by labeled Envision anti-rabbit System (Dako, Glostrup, Denmark), were used for anti-aldolase-A and anti-Grp94, whereas the labeled Envision anti-rabbit System alone was used for antiactinin 4. Sections stained for anti-VCP were incubated with

anti-mouse IgG (Vector Laboratories), followed by the avidin–biotin amplification method (Vectastain, Vector Laboratories). The immunoreaction was revealed by horseradish peroxidase using 3,3′-diaminobenzidine (Biogenex, San Ramon, CA). The slides were slightly counterstained with Harris’s hematoxylin and analyzed with optical microscope Axioskope 2 (Zeiss, Gottingen, Germany), and images were acquired with AxioVision 4.4 system (Zeiss).

230

Results

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Serological Proteome Analysis of Colon Carcinoma and Tumor-Related Antigen Identification. Because we were looking for immunorecognized antigens shared by a significant number of patients, we decided to perform the serological screening on a colon carcinoma cell line rather than autologous tumor. This also permits us to have a reproducible and standardized assay and to overcome the heterogeneity of tumor tissue samples, as well as to overcome the necessity to confirm protein spot identification on each different sample.

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Journal of Proteome Research • Vol. 7, No. 2, 2008 507

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Figure 1. Proteome profile of the LS180 cell line resolved by 2DE after colloidal Coomassie Blue staining is shown on the left. A-D rectangles indicate regions containing colon-cancer-related protein spots resulting in the relevance of the serological analysis. The enlarged A-D regions (Coomassie Blue) and the corresponding reactivity of representative sera on LS180, HT29, and LoVo colon carcinoma cell lines, respectively (WB: patient sera), are shown (panels on the right). Arrows indicate the spots excised for MS analysis.

Table 3. Protein Identification by MALDI-TOF MS Analysis of Colon-Cancer-Related Immunoreactive Spots pI/Mra spot

1 2 3 4 5 6 7 8 9 10 11

protein name

glucose-regulated protein 94 R actinin 4 valosin-containing protein lamin A/C lamin A/C aldolase A aldolase A aldolase A heterogeneous nuclear ribonucleoprotein AB heterogeneous nuclear ribonucleoprotein AB heterogeneous nuclear ribonucleoprotein AB

accession number

experimental

theoretical

match. pep.c

seq. cov.d (%)

Mascot score

15010550 3157976 6005942 5031875 5031875 4557305 4557305 4557305 12803583

5.32/91509 5.39/90566 5.23/87264 6.24/63542 6.34/63542 6.91/36811 7.26/36580 7.58/36811 5.81/35959

4.73/90193 5.47/105224 5.14/89321 6.4/65131 6.4/65131 8.30/39420 8.30/39420 8.30/39420 9.04/36613

11 11 10 14 38 16 23 19 17

14 15 17 27 56 43 69 51 49

89 84 95 373 251 402 193 271 227

gi 12803583

6.10/36059

9.04/36613

9

23

76

gi 12803583

6.34/35859

9.04/36613

14

55

114

gi gi gi gi gi gi gi gi gi

a pI/Mr ) isoelectric point/relative molecular mass. sequence coverage.

248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269

b

b

Accession number ) NCBI protein database.

Total proteins extracted from the LS180 colon carcinoma cell line were separated by 2D-PAGE and transferred onto nitrocellulose membranes. Sera from 52 patients with colorectal cancer, 9 patients with colon adenoma, and 40 controls were screened individually for the presence of antibodies to colon carcinoma proteins (Tables 1 and 2). The image analysis of the immunoreactivity patterns obtained identified 11 protein spots recognized by patient sera with statistical significance compared to control sera. A similar pattern of reactivity was observed using HT29 and Lovo colon carcinoma cell lines, with the exception of spots 9–11 on HT29 (Figure 1). These protein spots were excised from a preparative gel for MALDI-TOF MS analysis (Figure 1), leading to the identification of 6 different proteins (Table 3). The specific immunoreactivity of colon carcinoma patient sera have a frequency ranging between 19 and 35% of the tumoral sera (Table 4a). Interestingly, antigens recognized with low frequency by patient sera showed high specificity not being or seldom recognized by control sera (Table 4a). The immunoreactivity against heterogeneous nuclear ribonucleoprotein AB (hnRNP AB) obtained with sera from suspected carcinoma patients subsequently diagnosed as adenomas was found to be statistically significant even when 508


c

match. pep. ) matching peptides.

d

seq. cov. )

considering the limited number of cases (n ) 9) (parts a and b of Table 4). The reactivity against hnRNP AB was maintained at less statistical significance in patients with tumor at early stages (I and II) of TNM classification21 (Table 1 and parts a and b of Table 4). These stages include tumors with different extension but without lymph node involvement (N0) or distant metastasis. The reactivity against hnRNP AB in patients with tumor at later stages (III and IV) characterized by metastasis in regional lymph nodes or distant organs (N1,2, M0,1 of TNM classification, respectively) was no more significant (Table 1 and parts a and b of Table 4). Interestingly, the reactivity against glucose-regulated protein 94 (GRP94), lamin C (LamC), and R-actinin 4 (Act4) became statistically significant or increased the statistical significance when considering only tumors at early stages (I and II), while the reactivity against valosincontaining protein (VCP) showed a higher statistical significance when considering the subgroup of tumors at late stages (III and IV) (Table 1 and parts a and b of Table 4). Aldolase A (AldA) was recognized by patient sera regardless of the disease stage (Table 1 and parts a and b of Table 4). Immunoreactivity in control sera might reflect the advanced age of the group because it is known that the presence of autoreactive antibodies

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Table 4. (a) Occurrence of Autoantibodies to the Immunoreactive Proteins in Colon-Carcinoma, Adenoma Patients and Control Subjects, (b) Immunoreactivity Pattern of Recognition in Colon Carcinoma at Different Stages of Disease Progression, and (c) Sensitivity and Specificity of the Serological Reactivitya a

positive sera

Fisher’s test

frequency (%)

protein

Ad

Cr (stg.I-II; stg.III-IV)

Cn

Ad versus Cn

Cr versus Cn

stg.I-II versus Cn

stg.III-IV versus Cn

Ad

Cr

Cr stg.I-II

Cr stg.III-IV

Cn

GRP94 Act4 VCP LamC AldA hnRNP AB

2/9 1/9 1/9 3/9 4/9 4/9

10/52 (8/25; 2/27) 10/52 (6/25; 4/27) 10/52 (4/25; 6/27) 18/52 (10/25; 8/27) 18/52 (8/25; 10/27) 11/52 (6/25; 5/27)

2/40 -/40 -/40 4/40 4/40 2/40

ns ns ns ns p ) 0.028 p ) 0.007

ns p ) 0.004 p ) 0.004 p ) 0.007 p ) 0.007 p ) 0.035

p ) 0.005 p ) 0.002 p ) 0.019 p ) 0.006 p ) 0.046 p ) 0.047

ns p ) 0.023 p ) 0.003 ns p ) 0.013 ns

22 11 11 33 44 44

19 19 19 35 35 21

32 24 16 40 32 24

7 15 22 30 37 19

5 0 0 10 10 5

b

sensitivity (%) c

hnRNP AB GRP94 LamC Act4 VCP AldA all TAAs

Cr

stg.I-II

stg.III-IV

specificity (%)

21 19 35 19 19 35 78.8

34 32 40 24 16 32 84

19 7 30 15 22 37 74

95 95 90 100 100 90 75

a GRP94, glucose-regulated protein 94; Act4, R actinin 4; VCP, valosin-containing protein; LamC, lamin C; AldA, aldolase A; hnRNP AB, heterogeneous nuclear ribonucleoprotein AB; Cr, colon cancer (n ) 52); stg.I-II, colon cancer with no regional lymph node metastasis (n ) 25); stg.III-IV, colon cancer with regional lymph node metastasis (n ) 27); Ad, adenoma (n ) 9); Cn, control (n ) 40); p, statistical significance; ns, not significant.

292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

increases with aging29 and did not correlate with nontumoral pathologies (Table 2). The overall immunoreactivity for the 6 identified proteins covers 78.8% of the colon carcinoma population tested. In fact, 41 of 52 patient sera were reactive with at least one of the relevant protein spots (Table 1). Sensitivity and specificity of the serological reactivity against each of the putative TAAs have been evaluated by ROC curve methodology. Each of the TAAs could differentiate tumor patients from controls, with sensitivity and specificity ranging from 19 to 35% and 90–100%, respectively (Table 4c). If early and late tumor stages were considered separately, the sensitivity of hnRNP AB, GRP94, LamC, and Act4 increased in stage I-II, while the sensitivity of VCP and AldA increased in stage III-IV (Table 4c). The combination of all of the markers increased the sensitivity up to 78.8% for the entire colon cancer group, with specificity reduction of 75%, suggesting that these are independent markers. An optimal ratio between sensitivity and specificity is achieved, grouping reactivity against hnRNP AB, GRP94, and LamC for stage I-II (64% sensitivity and 85% specificity) and reactivity against Act4, VCP, and AldA for stage III-IV (55% sensitivity and 90% specificity). Antibodies from Colon Cancer Patients Recognize Specific Isoforms of Target TAAs. To confirm the protein identification, WB was performed using specific antibodies on LS180 proteome (Figure 2). In the case of AldA and hnRNP AB, the immunoreactivity of colon cancer patient sera was super-

Figure 2. Comparison between patient sera (right panels) and specific commercial antibodies (Ab) (left panels) reactivity. Western blot analysis on 2D maps obtained from LS180 cell lysates was performed. Arrows on the right panels indicate protein spots recognized by patient sera and identified by MS, while arrows on the left panels indicate the corresponding spots recognized by specific antibodies. To discriminate VCP and R-actinin 4 reactivity, the same nitrocellulose was sequentially incubated with respective commercial antibodies; the black arrows indicate the reactivity against R-actinin 4 obtained, following the anti-VCP reaction (white arrow). Brackets indicate the superimposable reactivity pattern between patient sera and specific commercial antibodies observed for aldolase A and hnRNP AB. Journal of Proteome Research • Vol. 7, No. 2, 2008 509


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Table 5. Antigen Expression Level, Presence of the Isoform(s) Recognized by Colon Carcinoma Sera, and Immunoreactivity of the Colon Carcinoma Patient Sera on Different Tumor Cell Lines Proteome CRC8 GRP94 exp

CC LS180 HT29 Lovo NSCLC Med Dem Calu-1 RCC Car Pre BAC MCF7 DAl MDA-MB-231 PAC PAC-44 CFPAC-1 A8184

Act4

iso

S

exp

iso

S

exp

h h h

+ + +

+ + +

h h h

+ + +

+ + +

h h h

l l h

+

+

l l l

+ + +

l l l

-

l l

-

l l

+ +

l h

-

h h l

+ + -

l l l

+ + +

h l l

l l l

-

h l l

+ + +

h h l

+ +

CRC23

VCP

LamC

iso

S

exp

+ + +

+ + +

h h h

hnRNP

AldA

iso

S

exp

iso

S

exp

iso

S

+ + +

+ + +

l nd l

+

+

+

+

h h h

+ + +

+ + +

h h nd

+ + -

+ +

l l nd

+ +

+ +

h h h

+ + +

+ + +

h l

+ -

+

l nd

+

+

h h

+ +

+ +

+ + -

h nd nd

+ -

+

nd nd nd

h l l

+ + +

+

-

h nd h

+ +

+

nd nd nd

h h h

+ + +

+ + +

+

+

+

a CC, colon carcinoma cell lines; NSCLC, nonsmall cell lung carcinoma cell lines; RCC, renal cell carcinoma cell lines; BAC, breast adenocarcinoma cell lines; PAC, pancreatic adenocarcinoma cell line; GRP94, glucose-regulated protein 94; Act4, R actinin 4; VCP, valosin-containing protein; LamC, lamin C; AldA, aldolase A; hnRNP AB, heterogeneous nuclear ribonucleoprotein AB; exp, antigen expression level as detected by commercial Ab; h, high expression; l, low expression; nd, not detectable; iso, presence of antigen isoforms recognized by colon carcinoma sera as detected by commercial Ab; S, serological reactivity of colon carcinoma patient sera CRC8 and CRC23.

318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351

imposable to that of commercial antibodies (brackets in Figure 2), indicating that all of the different isoforms of these proteins were recognized. Unlike commercially available antisera that reacted with all isoforms of GRP94, Act4, VCP, and LamA/C, colon cancer sera recognized only some isoforms (arrows in Figure 2). This suggested that only some specific isoforms of the identified proteins became immunogenic in colon cancer patients. Reactivity of Antibodies from Colon Carcinoma Patients on Different Tumors. Two colon carcinoma patient sera were selected for their reactivity covering all of the six identified proteins (CRC8 for GRP94, Act4, VCP, LamA/C, and hnRNP AB; CRC23 for AldA) and used to screen the proteome resolved by 2DE of different colon carcinoma cell lines and cell lines belonging to different types of cancer. The reactivity against the identified proteins was confirmed on the HT29 and Lovo colon carcinoma cell lines, except for hnRNP AB on HT29 (Figure 1 and Table 5). Nonsmall cell lung carcinoma (NSCLC), renal cell carcinoma (RCC), breast adenocarcinoma (BAC), and pancreas ductal adenocarcinoma (PAC) cell lines were also analyzed. In most of the cell lines, colon patient sera were reactive against AldA and LamC, suggesting that these antigens were shared among different tumors. In noncolon cell lines, scattered reactivity was observed against GRP94 and hnRNP AB. Reactivity against VCP was observed only in one RCC cell line, while no reactivity was observed for Act4 (Table 5). This suggested a possible colon cancer specificity of the elicited immune response against these two antigens. Expression level or the presence of specific isoforms of the relevant antigens in the cell lines was compared to the serological reactivity (Table 5). A high level of protein expression and the presence of the isoforms recognized by colon cancer patients sera correlate with the serological reactivity for the GRP94, Act4, and AldA antigens, with the unique exception 510


Table 6. Reactivity of Sera from Patients with Different Tumors on the LS180 Proteomea PACs (n ) 23) LCs (n ) 9)

GRP94

Act4

VCP

LamC

AldA

hnRNP AB

0/23 0/9

0/23 0/9

0/23 0/9

0/23 6/9b

3/23 3/9

0/23 0/9

a PACs, pancreatic ductal adenocarcinoma sera; LCs, lung carcinoma sera. b Statistical significance as assessed by Fisher’s test (p < 0.001).

of Act4 in PAC-44 cell line, in which, despite the high expression level, the protein was not recognized by sera. In the case of LamC and hnRNP antigens, serological reactivity was associated with protein expression, even with low detectability as for hnRNP. While in the case of VCP, the serological reactivity depended upon the presence of the isoform recognized by colon cancer sera, with the exception of the MCF7 cell line. Reactivity of Sera from Pancreas and Lung Cancer Patients on the Colon-Cancer-Derived Proteome. To verify whether antibody responses specific for the 6 antigens identified in colon cancer could also occur in patients with other types of tumors, sera from 23 pancreatic carcinoma and 9 lung carcinoma patients were analyzed for their reactivity against the colon carcinoma cell line LS180 proteome. Sera reactivity, although not statistically significant compared to controls, was observed against AldA in both pancreatic (3 of 23) and lung (3 of 9) cancer patients. In contrast, a statistically significant frequency of lung carcinoma sera (6 of 9, p < 0.001 by Fisher’s test) reacted with LamC (Table 6). Tumor-Related Protein Antigen Expression on Primary Tumor Tissues. To evaluate the level of expression in primary tumors of the proteins found to be immunogenic in the SERPA, we analyzed colon cancer surgical specimens by WB using specific antisera. Figure 3A shows the reactivity observed on tumoral and adjacent normal mucosa tissues of three patients. AldA and Act4 proteins were detected exclusively or found to

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Figure 3. Expression of the identified cancer-related proteins in tumor and normal mucosa. (A) Proteins obtained from transformed and normal tissue isolated from patients were resolved by SDS-PAGE and transferred to nitrocellulose. Protein expression was assessed by WB performed with specific antibodies on three (CRC36, CRC30, and CRC12) patient specimens. Arrows indicate the reactivity corresponding to GRP94 and the reactivity against the Lamin isoforms A and C. T, tumoral tissue; N, normal mucosa. Reactivity with anti-β-actin mAb (lower panels) was used as a protein-loading control. (B) Immunodetection of the colon-cancer-related proteins on matched primary tumor and normal mucosa sections obtained from representative patients of the colon carcinoma cohort (n ) 16) analysed for serological reactivity. Normal mucosa (a-d) and its tumor counterpart (a′-d′) were stained with specific antibodies (anti-GRP94, a and a′; anti-R-actinin 4, b and b′; anti-VCP, c and c′; and anti-aldolase A, d and d′). Black arrows indicate reactive cells. Rectangles in c and c′ show enlarged images, highlighting different intracellular reactivity for VCP staining, mainly nuclear in normal tissue and both nuclear and cytoplasmic in tumoral specimens. White arrows indicate the nuclei. N, colonic normal mucosa; T tumoral tissue. Magnification 20×. (C) Graphic representation of the immunohistochemistry analysis performed on colon tumoral and normal tissue samples obtained from patients (n ) 16) at the time of sera collection. On the y axis is reported the number of patients corresponding to quantitative and qualitative staining evaluation. percentage of stained epithelia in sections: 0, no stained cells; 1, up to 10% of the transformed or normal epithelial cells; 2, 10-50% of the transformed or normal epithelial cells; and 3, more than 50% of the transformed or normal epithelial cells. Staining intensity: 0, no signal; 1, weak; 2, moderate; and 3, intense. T: black bars, tumoral tissue. N: white bars, normal mucosa.

378 379 380 381 382 383 384 385

have an increased expression in tumor compared to its normal counterpart. GRP94 and LamA/C showed in tumor either overor equal expression compared to normal tissue. Interestingly, the LamA/C expression increase was mainly due to isoform C (represented by the lower immunoreactive band in Figure 3A, see arrow). VCP was highly expressed both in tumoral and normal specimens, while hnRNP AB was barely detected in both.

Although suggestive of protein expression levels in tumor, the WB reactivity may also be affected by protein expression in nontumoral cells present in the lysates. To better characterize the tumor-specific expression of the selected proteins, immunohistochemistry was performed on paired samples of tumoral and normal tissues. A total of 16 specimens collected from the patient group used for the immunoproteomic study were analyzed. This analysis confirmed the results obtained by Journal of Proteome Research • Vol. 7, No. 2, 2008 511

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Table 7. Immunohistochemistry on Tissue Samples from Colon Cancer Patients at the Time of Sera Collection and Serological Reactivity on the LS180 Cell Line Proteomea GRP94 patient number

23 30 49 12 48 31 32 14 34 35 25 37 18 46 11 29

Tb e

3–3 3–2 1–2 3–2 3–2 3–3 3–2 2–2 3–3 2–2 3–3 3–3 3–2 2–2 3–3 3–3

Act4

VCP

AldA

Nc

Sd

T

N

S

T

N

S

T

N

S

3–1 2–1 1–1 3–2 3–1 nd 2–1 1–1 0–0 nd 3–3 1–3 1–1 nd 1–1 0–0

+ + -

2–2 2–1 3–3 3–2 nd 1–1 3–3 1–1 1–1 1–2 3–2 1–1 2–3 1–1 3–2 2–2

0–0 1–1 0–0 0–0 nd nd 2–1 0–0 0–0 nd 0–0 0–0 0–0 nd 1–1 0–0

+ + + -

3–3 3–3 3–2 3–3 3–2 3–3 3–3 3–3 3–3 3–2 3–3 3–3 3–3 3–2 3–3 3–3

3–3 3–2 1–1 2–3 3–2 nd 3–2 1–1 2–2 nd 3–3 2–2 1–1 nd 1–1 1–2

+ +

2–2 2–1 3–2 0–0 1–1 2–2 3–2 2–2 3–3 1–1 3–2 2–3 2–1 2–1 2–3 3–2

0–0 0–0 0–0 0–0 0–0 nd 2–1 1–1 1–1 nd 3–2 0–0 0–0 nd 1–1 0–0

+ + + + + + -

a GRP94, glucose-regulated protein 94; Act4, R actinin 4; VCP, valosin-containing protein; AldA, aldolase A. b T ) tumoral tissue. c N ) normal mucosa. S, serological reactivity; nd, not determined. e First value: percent of stained epithelial component. 0, no stained cells; 1, up to 10% of the transformed or normal epithelial cells; 2, 10-50% of the transformed or normal epithelial cells; and 3, more than 50% of the transformed or normal epithelial cells. Second value: staining intensity. 0, no intensity; 1, weak; 2, moderate; and 3, intense. d

394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411

WB, permitting the topographic localization of the signal. The expression of GRP94, Act4, and AldA proteins was mainly present or found increased on neoplastic cells (parts B and C of Figure 3 and Table 7). The discrepancy observed in patient 12, in which AldA expression by WB was not confirmed by immunohistochemistry, may reflect a limit of the antibody sensitivity in the latter technique. VCP was found to be generally expressed on both tumoral and normal mucosa, even if immunostaining was found increased in 10 of 16 tumor samples (parts B and C of Figure 3 and Table 7). Interestingly, the VCP staining was mainly localized in the nucleus in normal epithelia while segregated in both the nucleus and cytoplasm in cancer cells (c versus c′ enlarged frame in Figure 3B). The antibody specific for the hnRNP AB failed to react in immunohistochemistry analysis. Because the reactivity against the LamC isoform is not distinguishable from that of LamA by the commercially available antibodies, data on its expression level and localization were not informative (data not shown).

412

Discussion

413

Six proteins were identified by serological proteome analysis to colorectal carcinoma as cancer-related antigens recognized by patient autoantibodies. These proteins have been previously described upregulated or aberrantly expressed in different tumoral tissues,30–37 but, out of AldA,38 a humoral immune response against them has not been reported thus far. Not much is known as to whether the antigenic pattern recognized by antitumor antibodies is modified during the progression of the disease. We have addressed this issue characterizing the antibody responses in patients with colorectal cancer at different stages of disease. Our analysis indicated a modification in antigen recognition by patients B cells as a function of colon cancer progression. Whether this reflects changes in antigenic properties of the tumor or changes in the efficiency of immune response is presently not known. Noteworthy, the results obtained by the use of sera from adenoma patients with a high grade of dysplasia, considered preneoplastic lesions, suggest that an immune response already occurs against pretumor cells. Our

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512


observations support the evidence that the humoral immune response occurrs preferentially at the early stages of colon carcinoma development.3 The immune recognition of hnRNP AB prevalently in patients with adenoma and in those with early stage tumors suggests an association of this nuclear matrix protein with early transformation events. In fact, rearrangement of nuclear matrix components, including a hnRNP AB expression increase, was found to be a feature of the early proliferative phases of colon and lung carcinomas.36,37,39–41 Interestingly, another protein constituent of the nuclear lamina, LamC, became immunogenic in colon carcinoma at early stages (I and II). At the same stages, patient sera recognized GRP94, a protein involved in innate and T-dependent antitumor immunological activity.42,43 GRP94 as well as AldA, which is recognized by patient sera regardless of the disease stage, are proteins related to the glycolytic metabolic pathway. Their overexpression, observed in this study and previously reported in colon and lung carcinomas,30,36,38 can be explained as a protective reaction of the tumor cell to survive glucose deprivation and hypoxia.44,45 An immune response restricted to the colon cancer cohort of patients was observed in patients at advanced stages (III and IV). Reactivity elicited by VCP and Act4, proteins having a role in protein degradation and cytoskeletal rearrangement, respectively, was able to discriminate between cancer and control individuals. A poor prognosis has been correlated with VCP overexpression in hepatocellular and gastric carcinomas and Act4 upregulation in breast cancer.31–33 The interesting trend of the changes of immunoreactivity along tumor progression might suggest that the major transformation events (nuclear rearrangement, increase of energy metabolism, cytoskeletal modifications, and protein degradation) can be tagged by the immunological system.

431

The immunoreactivity against AldA and LamC proteins reported in both lung and colon carcinoma sera (ref 38 and this work) suggested a shared pattern of immune recognition. However, this is not a general feature occurring in any tumoral transformation because sera from pancreas carcinoma patients failed to react with these antigens.

462

432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461

463 464 465 466 467


Serological Immunoreactivity against Colon Cancer Proteome 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530

The majority of the identified tumor-related antigens eliciting a humoral immune response described in the literature are differentiation antigens or overexpressed proteins.46 The association between protein overexpression and antibody response in patients with lung cancer has been reported, indicating upregulation as the basis for the immunogenicity.47 Our data support this hypothesis, because the patients that recognized one of the identified proteins did present either de novo or overexpressed the same protein in the tumor tissue. The humoral response against AldA in some colon cancer patients might reflect a specific over- or de novo expression by transformed intestinal epithelia. In fact, AldA isoform is expressed in the developing embryo, but its expression is normally repressed in the adult intestine. In the case of lamins A and C, alternatively spliced isoforms of the same gene, an increased expression of LamC compared to LamA was reported in lung carcinoma cell lines (ref 48 and this work, data not shown). Interestingly, a similar increased expression of the LamC isoform was observed in 2 of 3 tested colon primary tumors. Whether LamC upregulation might be a commune feature of colon cancer should be validated by comparing a larger panel of tumor and normal tissues. The absence of colon carcinoma sera reactivity on tumor cell lines of different origin mostly correlated with no or low protein detection, supporting the hypothesis that in colon carcinoma these proteins are overexpressed. However, not all of the patients presenting overexpressed proteins were found to develop a humoral response (Table 7).16,46 Thus, the expression level of the antigen seems to be an important feature but not sufficient to induce the antibody response. Another possible explanation for the protein immunogenicity is the presence of unusual isoforms as suggested by the immunoreactivity restricted to specific isoforms in the case of GRP94, Act4, VCP, and LamC. What features make these isoforms immunogenic is not known at the moment: different post-translational modifications occurring during tumoral transformation, different types of protein processing, and variability among tumors in the expression of MHC molecules and antigen presentation may all be responsible for immunogenicity of isoforms. Some mechanisms that allow cytoplasmic- and nuclearderived peptides to be recognized by MHC class II-restricted CD4+ T cells, the main lymphocyte population that helps B cell response, have been described.49 Among these, the chaperon-mediated autophagy is enhanced in tumor development.50 Accordingly, peptides belonging to VCP and hnRNP A2/B1 were eluted from tumor-surface-expressed MHC class II molecules.50,51 An alternative source of intracellular antigens is represented by the exosomes, small vesicles shed by tumoral cells that can have a role in triggering the immune response against cancer. Interestingly, Act4 has been identified in exosomes isolated from mesothelioma cells.52 Because only a subset of patients developed a humoral response to a specific antigen, the final goal should be the identification of a panel of antigens able to define the entire population of colon cancer patients. Even good specificity of a single marker does not show sufficient sensitivity, while, if used together, the putative markers increased the sensitivity with acceptable specificity. This underlines the idea that a panel of markers is needed for a multifactorial disease, such as colon cancer. From the clinical point of view, the relevance of having antibodies against the tumor-related proteins is not presently clear but their presence witness an immunological reaction that occurred during the cell transformation. The identification of new tumor-related antigens in colon cancer will lead to

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advances in cancer research by making tools available for the study of the natural immune response of patients against their own tumor. From this point of view, we are currently looking for a T-cell-specific response against the tumor-related antigens identified in colon carcinoma patients.

531

Acknowledgment. This work was supported by AIRC58p and 63D, Fondazione CARIPLO Nobel, and Lega Italiana per la Lotta Contro Tumori. P.D. and M.P.P. are supported by Compagnia di San Paolo and Fondazione Guido Berlucchi. We acknowledge Michael John for revising the manuscript, Franco Novelli (University of Torino) for helpful discussion, and Graziella Santambrogio for assistance in immunohistochemistry.

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PR070360M

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Serological Immunoreactivity against Colon Cancer Proteome Varies

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