Originated from the epithelial cells of the thymus, thymoma is one of the most common tumors of the anterior mediastinum.1 Thymoma is closely bound up with the neuromuscular disorder myasthenia gravis.2 Until now, the most widely used histopathological classification system for thymoma is the World Health Organization (WHO) histological classification, which was based on the Muller-Hermelink classification model.3,4
In light of epithelial cell types and the ratio of epithelial to lymphocytic cells, thymoma can be divided into four major types. Type A corresponds to spindle neoplastic oval cells. Type B corresponds to round epithelial cells with atypia or lymphocytes. Type AB contains a mixture of these two types. Type C thymoma is regarded as thymic carcinoma. Type B is further sub-classified into three subtypes, B1, B2 and B3, according to the increasing proportion of epithelial cells and the emergence of atypia.5
It is already clear that type C is a disparate group, and the diagnosis and treatment of this type of thymoma is relatively clear-cut. However, the distinction between B1, B2 and B3 is only made on the basis of the increasing proportion of epithelial cells and atypia. To some extent, it is more difficult to distinguish between types B1, B2, and B3 clearly, which was dependent on the pathologists' subjective judgment. However it is noteworthy that type B1 behaves more like a benign tumor, while types B2 and B3 are more aggressive. The prognosis for type B1 is better than for types B2 and B3 and the therapeutic measures used for different types are very different. An accurate distinction of thymoma B1 to B3 is very important for therapeutic decision making.
Recently, the molecular characteristics of the different histologic and clinical types of thymoma have been used to improve their diagnosis, therapy, and to determine the patients prognosis.2 Searching for biomarkers and tumor profiling for thymoma has been carried out in recent years using DNA microarray, but alterations in the proteome may reflect cellular changes more accurately since proteins are the actual mediators of intracellular processes as opposed to mRNA.6 A lack of study of the proteomic profiles of thymoma hinders our understanding of and cures for this tumor. The recent application of two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) to the study of tumors allows the characterization of alterations in protein expression that correlate with the thymoma classification.
In this study, we compared type B1 with type B2 thymoma tissues by 2-DE and MALDI-TOF-MS/MS. Some of the differentially expressed proteins were confirmed by immunohistochemistry. The relationship of their expression with clinicopathological parameters was investigated.
General chemicals such as acrylamide, and Tris buffer were purchased from Sigma Chemical (USA). 2-DE chemicals were obtained from GE Healthcare (USA). A monoclonal antibody to ezrin was the product of Abcam (USA). A polyclonal antibody to glutathione S-transferase pi (GSTP1) was bought from Santa Cruz Biotechnology.
Patients and tissues
Tumors from cases of thymoma that had undergone curative resection were randomly collected from the Department of Thoracic Surgery, Shanghai Chest Hospital. This study was approved by the Research Ethics Committee of Shanghai Jiao Tong University School of Medicine. All thymoma samples were obtained with informed consent from patients at the Shanghai Chest Hospital. None of the thymoma patients received radiotherapy and chemotherapy before surgery. The data for the patients included in this study are detailed in Table 1 and Table 2.
2-DE and MS analysis
Six samples of type B1 and six samples of type B2 thymoma tissues were sliced and washed three times with PBS to remove blood. The protein from a total of 100 mg of tissue was extracted by homogenization in 1 ml of lysis buffer (20 mmol/L Tris, 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 65 mmol/L DTT, 1 mmol/L PMSF, and 2% pharmalyte). The lysate was centrifuged at 20 000 r/min for 60 minutes at 4°C. The protein concentration was determined by the Bradford method. 2-DE was carried out according to the 2-DE manufacturer, Amersham Biosciences. A total of 400 μg of protein was loaded onto a 13 cm non-linear IPG strip (pH 3-10, GE Healthcare, Piscataway, NJ) for first-dimensional isoelectric focusing. Protein separation in the second dimension SDS-PAGE was carried out on Hofer SE600 system. After electrophoresis, the gels were stained with Coomassie brilliant blue (CBB) and scanned by an Imagescanner (GE Healthcare).
Selected protein spots were excised from 2-DE gels and destained with destaining solution (50% acetonitrile, 5 mmol/L NH4HCO3). In-gel digestion was performed with 30 ng of trypsin (Promega, USA) in 25 mmol/L ammonium bicarbonate for 15 hours at 37°C. The tryptic peptides were extracted from the gel with 0.2% TFA three times and dried with N2. Peptides were eluted with 0.1% TFA and mixed at 1:1 with 20 mg/ml a-cyano-4-hydroxy cinnamic acid (CHCA) in acetonitrile before spotting onto a stainless steel MALDI target plate. Spectra were obtained using a mass spectrometer MALDI-TOF-MS/MS (ABI 4700). Data were searched with the search engine MASCOT (Matrix Science Inc., USA) against an NCBI protein sequence database. The mass tolerance was set as 0.3 Da, and MS/MS tolerance was 0.4 Da. The automatic data analysis and database searching were performed by GPS Explore software.
Tissue array and immunohistochemistry
To prepare for a multitissue array, archival paraffin blocks from thymoma tumors from 69 patients were used. Representative areas of thymoma were selected carefully by a pathologist from hematoxylin and eosin-stained sections under a light microscope and were marked on individual paraffin blocks. Tissue array was performed using a TCW600 Manual Tissue Arrayer (Bonan Biotechnology, Shanghai, China) with a 2 mm diameter tissue core. The cores were then transferred into ready-made recipient blocks. The tissue arrays were cut into 3 μm sections and placed on slides. The tissue arrays were deparaffinized in four consecutive, 10-minute incubations in xylene, dipped into serial grades of ethanol to remove the xylene, followed by rehydrated for 5 minutes.
Antigen retrieval was carried out using 6.5 mmol/L citrate buffer (pH 6.0) pressure-cooking, and endogenous peroxidase activity was blocked with 2.5% hydrogen peroxide in methanol for 30 minutes at room temperature. The slides were incubated with the primary antibody against respective target proteins (Ezrin, 1:200; GSTP1, 1:100) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Immunocomplexes were detected with 3, 3-diaminobenzidine as the chromogen resulting in deposition of a brown reaction product. Samples were counterstained with hematoxylin, a blue nuclear stain. The cells containing brown granules were considered as positive. Five fields of cells (at least 100 cells totally) were counted at a magnification of ×200. The percentage of positive cells and the intensity of staining were assessed in a semi quantitative manner, and each tissue spot was given a total score based on the results. The percentage of positive cells was recorded by the following criteria: <5% for “0”; 5%-25% for “1”; 26%-50% for “2”; >50% for “3”. The intensity of staining was graded: absence or faint yellow for 0; moderate for 1; and strong for 2. Both percent positivity of cells and staining intensity were decided under double blind conditions. The final protein expression score was calculated with the value of percent positivity score×staining intensity score, which ranged from 0 to 6. Finally, the immunostaining of the tumor cells was assessed as negative (score 0-1), weakly positive (score 2-3), and strongly positive (score 4-6). The relationship of proteins' expression with clinicopathological parameters, such as tumor stage and WHO classification, were investigated using various statistical analysis.
Gel images were analyzed statistically by the Imagemaster 6.0 software package (GE Healthcare). The software uses standard criteria to automatically locate spots on gels, subtract levels of background intensity, and match detected spots within 2-DE gels. Spots with intensity changes greater than 2.0-fold were considered as differently expressed spots. The significance of a correlation between expression of ezrin and GSTP1 and the histological diagnosis was analyzed using the Mann-Whitney U test. Correlation between expression of each protein and the clinical stage, as well as the WHO classification, was estimated by Spearman's Rank Correlation Test. The SPSS 13.0 (SPSS Inc., USA) software package was used to perform statistical analyses. A P value of less than 0.05 was considered to be statistically significant.
Differences in protein expression between B1 and B2 thymoma tissues
To compare the global protein profiles between type B1 and B2 thymoma, we first did 2-DE gel electrophoresis analysis of total lysates prepared from twelve type B thymoma tissues. Following Coomassie brilliant blue (CBB) staining (Figure 1), the gels were subjected to Image Master imaging analysis. Protein spots that have differences of ≥2.0 between the two types of tissue in triplicate experiments were chosen for further analysis. Fifty such spots were found and they were subjected to MALDI-TOF-MS analysis. Twenty-four of these spots were identified with good peptide coverage, high Mascot scores, and similar observed calculated molecular weight and isoelectric point (Table 3, Figure 1). Figure 2 shows the PMF of protein spots 2, 10, 18, and 24 representing ezrin, GSTP1, CK18 and HSP-27, respectively. Among them, six spots were confirmed as plasma abundant proteins, such as beta-globin and serum albumin, and two spots were identified as the same protein. Finally, sixteen proteins from thymoma tissues were successfully identified.
Of the 16 protein spots identified, QPRTase, nucleophosmin (NPM), GSTP1, Apo-AI, methylglyoxalase, tubulin-specific chaperone A, RNA-binding motif protein 3, Heat shock cognate 71 kD, ezrin, fibrinogen gamma chain, hnRNP H, peroxiredoxin-6, triosephosphate isomerase, and eIF-5A2 were increased in expression in the type B2 thymoma tissues. The collagen alpha-1 chain, PDIA3, and keratin 19 were decreased in the type B2 thymoma tissues. hnRNP H was identified in two separate spots, suggesting the occurrence of post-translational modifications.
Correlation between ezrin or GSTP1 and WHO classification
Of all the proteins that have altered expression in type B thymoma tissues, ezrin and GSTP1, which were increased in type B2 thymoma tissues, were considered to be of interest because of their role in cell signal transduction. To determine if the increased expression of ezrin and GSTP1 potentially contributes to thymoma progression, we first did a correlation analysis of their expression with WHO classification.
The expression of GSTP1 and ezrin were confirmed by immunohistochemistry in a tissue array including 69 thymoma samples from B1 to B3 thymoma tissues that were not included in the 2-DE experiment (Figure 3). Results of immunohistochemistry for GSTP1 and ezrin in thymoma are summarized in Table 4 and Table 5. In positive cases, the tumor cells showed cell membrane and cytoplasmic staining for ezrin and showed cytoplasmic staining for GSTP1. The immunostaining of tumor cells for ezrin and GSTP1 were significantly stronger in the peripheral areas and invasive nests than in the central areas. In non-neoplastic thymic tissue adjacent to the thymoma, the epithelial cells were negative for GSTP1.
Immunoreactivity of ezrin was demonstrated in 1/8 type B1 cases (12.5%), 4/7 type B2 cases (57.1%), and 14/18 type B3 cases (71.4%). Statistically significant differences were observed in the positive rate of ezrin between type B1 thymoma and type B3 thymoma (Z= -2.963, P <0.01). However, there is no significant difference in ezrin expression between type B1 and B2 thymoma (Z= -1.814, P >0.05) or between type B2 and B3 (Z= -1.047, P >0.05). The ezrin expression showed a tendency to increase from WHO type B1 to type B3, Spearman's correlation coefficients 0.515, P <0.01. The expression rate of GSTP1 in type B1 thymoma was 5.6% (1/18). In type B2 thymoma tissues, GSTP1 had moderate expression, with rates of 43.8% (7/16). In type B3 thymoma tissues, GSTP1 was highly expressed, with expression rates of 80.0% (8/10). The statistical analysis demonstrated that type B2 and B3 groups had significantly higher positive expression of GSTP1 than the B1 group; type B2 vs. B1 Z= -2.582 and P ≤0.01, type B3 vs. B1 Z= -4.012 and P <0.001. A statistically significant difference was observed in the positive rate of GSTP1 between type B2 thymoma and type B3 thymoma (Z=-2.464 and P <0.05). Moreover, the results showed a high correlation existed between GSTP1 expression and WHO classification from type B1 to type B3 (Spearman's correlation coefficients 0.633, P <0.001). In all, these results suggest that the elevated expression of ezrin and GSTP1, especially GSTP1, can likely contribute to the discrimination of types B1, B2 and B3 thymoma tumors.
Correlation between ezrin or GSTP1 and clinical stage of thymoma
Next, we examined and compared the expression of ezrin and GSTP1 protein in different clinical stages of thymoma. The results are presented in Table 6 and Table 7. Of the 27 thymoma examined, 14 (51.9%) were positive for ezrin; 2/7 stage I thymoma, 3/8 stage II thymoma, and 9/12 stage III thymoma. The statistical analysis demonstrated that the stage III group had significantly higher positive expression of ezrin than the stages I or II groups; stage III vs. stage I Z= -2.210 and P <0.05, stage III vs. stage II Z= -2.098 and P <0.05. Of the 13 stage I thymoma tumors examined, only 1 specimen (7.6%) was positive for GSTP1 and of the 13 stage II thymoma examined, 3 specimen (23.1%) were positive for GSTP1. In contrast, 7/11 (63.6%) stage III thymoma tumors were positive for GSTP1. There is a significant difference in GSTP1 expression between Stage I and Stage III, Z= -2.862 and P <0.01.
Statistical analysis showed that there was a correlation between GSTP1 or ezrin expression and clinical stage; Spearman's correlation coefficient for Ezrin was 0.481, P <0.05 and for GSTP1 was 0.484, P <0.01. Taken together, the above results showed that the increased expression of ezrin and GSTP1, particularly GSTP1, were correlated with tumor stage.
Clinical and pathological heterogeneity of thymoma hinders selection of appropriate treatment for individual cases.3,7 Molecular profiling at gene or protein levels may elucidate the biological variability of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters.8 Several studies have explored the biologic behavior of thymic epithelial tumors, showing that expression of P53, CD117, bcl-2 and Ki-67 were associated with clinical stage and prognosis.9–11 Recently, on the basis of the WHO histological classification system, Takahashi et al12 found that some matrix metalloproteinases (MMPs) were characteristically expressed in certain thymoma types; MMP2 in type B3 (77.8%), MMP7 in type C (100%), and MMP9 in type B2 (100%). Moreover, expression of MMP2 and MMP7 showed a correlation with clinical stage. Although a number of previous reports related to thymoma classification were documented, so far, few proteomic studies have been done in thymoma research. Hence, in the present study, our goal was to use high throughput proteomics to help identify candidate biomarkers for WHO histologic classification type.
In this study, we used proteomics to determine the differential expression of proteins between type B1 and B2 thymoma tissues. We found 50 protein spots with significant difference in expression between these two types. Subtracting the redundant protein, sixteen proteins were successfully identified by MALDI-TOF MS. Two of them, ezrin and GSTP1, had commercially available antibodies and their protein expression in clinical samples was validated using immunohistochemistry. Their expression was proved to be closely correlated with clinical stage and WHO classification.
As a member of the ERM protein family, ezrin serves as an intermediate between the plasma membrane and the actin cytoskeleton.13 Functions of the ezrin include cell surface adhesion, migration, and organization as well as functioning as a signal transducer.13 It can convey an anti-apoptotic signal by activation of the PI 3-kinase/Akt pathway.14 It can also interact with several transmembrane adhesion molecules, such as CD44 and ICAM-1.14–17 Overexpression of ezrin promotes cell protrusion, anchorage-independent growth, motility and invasion in the pancreatic cancer cell line MiaPaCa-2.18 The siRNA-mediated downregulation of ezrin gene expression inhibits the migration and invasion of the human gastric cancer cell line SGC-7901, and improved cell adhesion as well as increased sensitivity to camptothecin-induced apoptosis.19 Significant association between ezrin over-expression was found with advancing histological grade in myxofibrosarcoma, colorectal cancer, and with poor outcome in primary osteosarcoma.20,21
GSTP1, a member of Glutathione S-transferases (GSTs) family, plays an important role in detoxification by catalyzing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione.22 Beyond that, it is involved in the regulation of cell proliferation and apoptosis by direct interactions with JNK.23 In the past few years, GSTP1 was found to be overexpressed in a variety of epithelial carcinomas, including gastrointestinal, esophageal carcinoma, lung cancer, and breast cancer.24,25 High GSTP1 levels are significantly associated with advancing tumor stage and poor outcome in ovarian cancer.26 Many articles24,25,27–29 have reported that GSTP1 genetic polymorphisms represent risk factors for colorectal cancer, gastric cancer, and bladder cancer. In addition, hypermethylation of GSTP1 is an early event in breast carcinogenesis.30 In particular, GSTP1 has also been shown to be over expressed, and contribute to chemotherapy resistance, in a series of chemotherapy resistant cell lines.31,32
In our studies, the clinicopathological comparison by tissue array reveals that ezrin and GSTP1 have higher expression in advancing histological grade thymoma type B3. Ezrin and GSTP1 were highly expressed in stage III tumors. The expression of ezrin and GSTP1 were strongly correlated with the malignant progress from type B1 to type B3. It also showed a correlation with clinical stage. All these findings are in accordance with those in other types of solid tumors. The results indicate that at least these two proteins may play important roles in the progression of thymic epithelial tumors, and they may be used as diagnostic markers for thymic tumor classifications; especially as biomarkers for distinguishing type B1 and type B2 thymoma in the clinic.
The other 14 differentially expressed proteins we identified are involved in protein synthesis and folding (HSCT1), cytoskeleton components (CK 19), toxin catabolism (PRDX6), and substance metabolism (QPRT, Methylglyoxalase, TIM). Some of them have been reported to be related to tumor progression or chemotherapy resistance; such as NPM, methylglyoxalase, and PRDX6. CFA is involved in the pathway leading to correctly folded beta-tubulin from folding intermediates and plays a role in capturing and stabilizing beta-tubulin intermediates in a quasi-native confirmation.33 Another important group comprises proteins involved in glycometabolism and drug metabolism, e.g. methylglyoxalase, TIM, PRDX6, and GSTP1. PRDX6 and GSTP1 are involved in protection against oxidative stress. They are widely implicated in the development of various cancers.22,34 Among the other proteins identified, we also found a pool of nucleoproteins which regulated RNA processing and DNA replication; e.g. NPM, RNA-binding motif protein 3, and hnRNP H. In common with GSTP1 and ezrin, most of the remaining protein spots are not reported to have direct roles in thymoma progression. It is possible that these proteins may be also involved in thymoma progression. Future studies will be concentrated on these remained proteins. It is noteworthy that none of the MMPs which have been associated with WHO classification were identified in this study. MMPs are extracellular proteins, which are unlikely to be extracted, separated and identified by common extraction methods, two-dimensional gel electrophoresis and MALDI-TOF. Clearly, the proteomic technique used in this study has inherent limitations.
To sum up, we have carried out the first proteomic analysis of human thymoma tissues, and more importantly, we have performed the study of the protein expression profiles in different histologic subtypes. We have identified a series of proteins with significantly altered abundance in type B1 and B2 thymoma tissues. We have for the first time identified a different expression pattern of ezrin and GSTP1 protein in thymic epithelial tumors classified by the WHO criteria. Our results indicate that this molecular classification system, based on the statistical analysis of ezrin and GSTP1 immunohistochemical profiling, is a useful approach for thymoma classification. The identification of specific protein signatures associated with the classification of subtypes may provide novel biomarkers that provide more accurate prognostic information and may help to identify new molecular therapeutic targets and clues for understanding the molecular mechanisms governing thymoma progression.
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