Elsevier

Journal of Proteomics

Volume 75, Issue 3, 4 January 2012, Pages 833-844
Journal of Proteomics

Proteomic study of malignant pleural mesothelioma by laser microdissection and two-dimensional difference gel electrophoresis identified cathepsin D as a novel candidate for a differential diagnosis biomarker

https://doi.org/10.1016/j.jprot.2011.09.026Get rights and content

Abstract

To investigate the proteomic background of malignancies of the pleura, we examined and compared the proteomic profile of malignant pleural mesothelioma (MPM)(10 cases), lung adenocarcinoma (11 cases), squamous cell carcinoma of the lung (13 cases), pleomorphic carcinoma of the lung (3 cases) and synovial sarcoma (6 cases). Cellular proteins were extracted from specific populations of tumor cells recovered by laser microdissection. The extracted proteins were labeled with CyDye DIGE Fluor saturation dyes and subjected to two-dimensional difference gel electrophoresis (2D-DIGE) using a large format electrophoresis device. Among 3875 protein spots observed, the intensity of 332 was significantly different (Wilcoxon p value less than 0.05) and with more than two-fold inter-sample-group average difference between the different histology groups. Among these 332, 282 were annotated by LC–MS/MS and included known biomarker proteins for MPM, such as calretinin, as well as proteins previously uncharacterized in MPM. Tissue microarray immunohistochemistry revealed that the expression of cathepsin D was lower in MPM than in lung adenocarcinoma (15% vs. 44% of cases respectively in immunohistochemistry). In conclusion, we examined the protein expression profile of MPM and other lung malignancies, and identified cathepsin D to distinguish MPM from most popular lung cancer such as lung adenocarcinoma.

Graphical abstract

Highlights

►We aimed to identify proteomic differences between lung malignancies on pleura. ►Laser microdissected tumor tissues were examined by 2D-DIGE. ►Comparative study identified proteins with differential expression. ►Western blotting and tissue microarray validated the proteomics results. ►Expression of cathepsin D was unique to lung adenocarcinoma.

Introduction

Malignant pleural mesothelioma (MPM) is considered an almost incurable tumor, showing resistance to chemo- and radio-therapy, and the median survival remains 8 to 18 months [1], [2]. Approximately 10,000 to 15,000 MPM cases are newly diagnosed annually worldwide. The incidence of the disease is rising, probably because its etiology is associated with prior exposure to asbestos, which has been widely used as construction material worldwide. Because of the lack of specific clinical symptoms as well as diagnostic biomarkers, early diagnosis and differential diagnosis between MPM and other pleural malignancies are often difficult [3], [4].

Global genetic studies have revealed the unique molecular background of MPM. Chromosomal aberrations including the frequent loss of chromosome 22 [5] and structural rearrangements of 1p, 3p, 9p and 6q [6], [7] were identified by karyotyping and comparative genomic hybridization analyses. Genetic studies revealed frequent inactivation, mutation and down-regulation of tumor suppressor genes such as p16 [8], p14 [9] and NF2-netrin [10]. Aberrantly methylated genes such as TMEM30B, KAZALD1 and MAPL13 were demonstrated by global methylation analyses for CpG islands [11], while transcriptome sequencing revealed mutations in XRCC6, PDZK1IP1, ACTR1A and AVEN in MPM [12]. These studies furthered our understanding of the genetic background of MPM and may lead to clinical applications that will improve the clinical outcome for MPM patients.

Previous studies have also shown that MPM is characterized by the immunohistochemical expression of epithelial membrane antigen, calretinin, WT1, cytokeratin 5/6, HBME-1 and mesothelin [3], [4]. Although these immunohistochemical biomarkers may help in the differential diagnosis of MPM to some degree, novel biomarkers with higher sensitivity and specificity have also been long required. Proteomic studies on MPM conducted to date have used tissue microarray samples [13], plasma samples, pleural effusions [14] and cell lines [15], [16], [17], [18], [19], none however, has used direct protein extracts from surgical specimens of MPM, probably because MPM has a complex histology and clinical materials are difficult to obtain.

In this paper, we report the proteins that distinguish MPM from the other lung and pleural malignancies, with the aim of developing differential diagnostic biomarkers. We compared the proteome of MPM with that of malignancies that may occur on the pleura and are therefore included in the differential diagnosis of MPM; these include lung adenocarcinoma, squamous cell carcinoma of the lung, pleomorphic carcinoma of the lung and synovial sarcoma. As the tumor tissues of these malignancies include various types of cells, laser microdissection was employed to recover specific tumor cell populations and thus allow accurate protein expression profiling. A combination of two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry was used to identify proteins unique to MPM tissues. Finally, we validated the expression of cathepsin D in MPM and lung adenocarcinoma using tissue microarrays.

Section snippets

Materials

Surgical specimens were obtained from patients with pleural malignancy, who were subjected to tumor resection with curative intent at the National Cancer Center Hospital between 1998 and 2007. Primary tumor tissues from MPM (10 cases), synovial sarcoma (6 cases), lung adenocarcinoma (11 cases), lung squamous cell carcinoma (13 cases), and lung pleomorphic carcinoma (three cases) were included in the study. The tumor tissues were frozen at − 80° at a time of pathological diagnosis, and embedded

Results

Modalities that will aid differential diagnosis continue to be in urgent need, and the identification of biomarkers for MPM may lead to such diagnostic modalities. As the proteome is a functional translation of the genome, regulating the malignant phenotypes of tumor cells, research strategies employing a proteomic approach are among the most promising for biomarker identification and development. However, there has been no report of successful protein expression profiling using direct protein

Discussion

We demonstrated that the combination of laser microdissection technique with 2D-DIGE, one of the most popular proteomic modalities, enabled the identification of proteins unique to histologically different lung cancers. Total number of protein species observed in this study was 3875, which may be quite large considering that the samples were obtained by laser microdissection. The approach in this study could be applicable to any types of malignant tumors. We also demonstrated the utilities of

Conclusions

Using surgically resected tissues and 2D-DIGE, we identified the proteins the expression of which is statistically significantly different between MPM and malignancies that may arise from the pleura. Our report demonstrated the advantages offered by the combined use of laser microdissection and 2D-DIGE with highly sensitive fluorescent dye for diseased tissues with complex histology. We also demonstrated the utilities of TMA for validation study. The identified proteins included established

Acknowledgments

This work was supported by a grant from the Ministry of Health, Labor and Welfare and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan. We appreciate Dr. Akihiko Yoshida for the useful pathology consultation.

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