Elsevier

Clinical Lung Cancer

Volume 15, Issue 5, September 2014, Pages 372-378.e1
Clinical Lung Cancer

Original Study
Identification of ENO1 As a Potential Sputum Biomarker for Early-Stage Lung Cancer by Shotgun Proteomics

https://doi.org/10.1016/j.cllc.2014.05.003Get rights and content

Abstract

Background

Lung cancer is the leading cancer killer. Early detection will reduce the related deaths. The objective of this study was to identify potential biomarkers for early-stage lung cancer in sputum supernatant.

Materials and Methods

Using shotgun proteomics, we detected changes in protein profiles that were associated with lung cancer by analyzing sputum supernatants from 6 patients with early-stage lung cancer and 5 cancer-free controls. Using western blotting, we validated the proteomic results in 22 lung cancer cases and 22 controls. Using enzyme-linked immunosorbent assay (ELISA), we evaluated the diagnostic performance of the biomarker candidates in an independent set of 35 cases and 36 controls.

Results

Proteomics identified 8 biomarker candidates for lung cancer. Western blotting validation of the candidates showed that enolase 1 (ENO1) displayed a higher expression level in patients with cancer than in cancer-free individuals (P = .015). ELISA revealed that the assessment of ENO1 expression in sputum supernatant had 58.33% sensitivity and 80.00% specificity in distinguishing patients with stage I lung cancer from cancer-free individuals.

Conclusion

The analysis of protein biomarkers in sputum may provide a potential approach for the early detection of lung cancer. Future validation of all the candidates defined by shotgun proteomics in a large cohort study may help develop additional biomarkers that can be added to ENO1 to provide more diagnostic efficacy for lung cancer.

Introduction

Lung cancer is responsible for 29% of all cancer deaths in men and women, causing more deaths than breast, colon, and prostate cancers combined.1 Approximately 85% of lung tumors are non–small-cell lung cancers (NSCLCs).2 NSCLC comprises 2 major histologic subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (AC).2 The development of easily performed noninvasive approaches for early detection of NSCLC followed by suitable treatments can reduce mortality.1, 3, 4

Sputum is one of the most noninvasively accessible body fluids. Numerous studies have shown that molecular genetic changes in the exfoliated respiratory epithelial cells of sputum could provide potential biomarkers for early-stage lung cancer.5, 6, 7, 8, 9, 10, 11 The exfoliated epithelial cells in sputum mainly comprise (1) bronchial epithelia that are derived from centrally located tumors that are mainly SCC, (2) respiratory epithelia that may share clonally molecular genetic lesions with SCC tumors but are not directly shed from the primary SCC tumors. Therefore, the bronchial epithelial cell-based analysis often provides higher accuracy for SCCs that are centrally located in the lungs compared with ACs that arise peripherally.5, 6, 7, 8, 9, 10, 11

Previous studies of the exfoliated epithelial cells in sputum have focused mainly on genetic and epigenetic analysis of nucleic acids to measure the sequence, copy number, mutation, methylation, and expression changes of genes.5, 6, 7, 8, 9, 10, 11 However, few of the genetic and epigenetic analytic approaches in sputum have been integrated into clinical practice and shown to have an impact on reducing the mortality of NSCLC. Proteins are the ultimate products of gene expression and are more diverse than DNA or RNA. Furthermore, alternative splicing and more than 100 unique posttranslational modifications from each gene can create 10s to 100s of species of protein.12 In addition, many physiologic changes are mediated posttranscriptionally and will not be revealed at the nucleic acid level.13 Moreover, proteins are more dynamic and reflective of cellular physiology and carry more information than nucleic acids. Therefore, the analysis of protein changes in sputum may provide an alternative means for diagnosis of early-stage lung cancer.

Differing from the epithelial cells exfoliated from local respiratory tract sites, sputum supernatant is a circulating cell-free body fluid, which may contain molecules originating from primary tumors either as a result of metastasizing cells or from leakage from the tumors into the circulation. The assessment of the circulating molecules, eg, proteins, in sputum supernatants may present a potential approach to help diagnose lung cancer, particularly ACs that are difficult to detect by studying exfoliated epithelial cells.

Mass spectrometry (MS)-based proteomics represents an important technological choice for arraying and characterizing constituent proteins. MS was used to characterize protein expression in bronchoalveolar lavage fluid from individuals with cystic fibrosis to discover potential biomarkers for the disease.14, 15, 16 Of the proteomic techniques, shotgun proteomics combining liquid chromatography (LC) and MS can globally delineate proteome profiles in complex mixtures and rapidly identify biomarker candidates in clinical samples.17

This study, which to our knowledge represents the first proteomic study using in-gel digestion coupled with LC/MS to address differential proteins of sputum supernatants in patients with lung cancer versus control individuals, aims to identify protein biomarkers in sputum that may potentially be useful in the early detection of the disease.

Section snippets

Patients and Sputum Collection and Preparation

Figure 1 shows the design for biomarker discovery and validation in this study, which was performed under a research protocol approved by the Institutional Review Board of University of Maryland Baltimore. As shown in Figure 1 and Table 1, a total of 64 patients with lung cancer and 64 cancer-free smokers were enrolled. In phase 1, we discovered significant changes of protein profiles that were associated with lung cancer by analyzing sputum supernatants from 6 patients with early-stage lung

Characteristics of Sputum Supernatant Proteins by Quantitative MS

To identify potential biomarkers for lung cancer, proteins of sputum supernatants from 6 patients with lung cancer and 5 healthy controls were separated by SDS-PAGE. As shown in Figure 2, all the samples had a major band around 50 kDa. The bands were then excised from all the samples and submitted for in-gel digestion and LC/MS analysis. After deconvolution of raw MS data, interrogation of the human protein database produced a total of 29 unique proteins (Supplemental Table 1 in the online

Discussion

Using 1D-GE and LC/MS-based shotgun proteomics, we separated protein changes in sputum supernatants and found that ENO1 displayed a high expression level in samples from patients with lung cancer. ENO1 is present on the surface of cells and plays an important role in tissue invasion and metastasis.29, 30 The enzymatic role of ENO1 is to convert 2-phosphoglycerate to phosphoenolpyruvate.29, 30 Activated ENO1 has a direct coupling to glycolytic activity.29, 30 Furthermore, ENO1 could be

Conclusion

Using shotgun proteomics together with western blotting and ELISA, we identify sputum ENO1 as a biomarker candidate for lung cancer. Although the 1-biomarker–based test is insufficiently discriminative to support undertaking a multicenter clinical trial, the findings from this study may lay the basis to perform a next step to develop multiple biomarkers that might have future clinical utility.

Disclosure

This work was supported in part by National Cancer Institute grant R01CA161837 and VA merit Award I01 CX000512, LUNGevity/Upstage Foundation Early Detection Award (FJ).

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