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DOI 10.1002/pmic.201300226

Proteomics 2014, 14, 186–195

RESEARCH ARTICLE

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma Yi-Jun Qi1 , Douglas G. Ward2 , Chun Pang3 , Qi-Ming Wang3 , Wenbin Wei2 , Jin Ma1 , Juan Zhang1 , Qiang Lou1 , Neil J. Shimwell2 , Ashley Martin2 , Nathalie Wong4 , Wei-Xia Chao1 , Ming Wang1 , Yuan-Fang Ma1∗ and Philip J. Johnson2 1

Key Laboratory of Cellular and Molecular Immunology, College of Medicine, Henan University, Kaifeng, P. R. China 2 School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK 3 Department of Clinical Oncology, Henan Tumor Hospital, Zhengzhou, P. R. China 4 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, SAR Hong Kong, China

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, “ConA-binding proCD” (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5–5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConApCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Received: June 10, 2013 Revised: October 2, 2013 Accepted: November 7, 2013

Keywords: Cathepsin D / Glycoproteins / Haptoglobin / Hepatocellular carcinoma / Human liver carboxylesterase 1



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Correspondence: Professor Philip J. Johnson, School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK E-mail: [email protected] Fax: +44121 414 3700 Abbreviations: CD, cathepsin D; ConA-pCD, ConA-binding pCD; GNA, snowdrop lectin; HCC, hepatocellular carcinoma; hCE1, hu-

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

Accounting for 500 000–1 000 000 new cases per year, hepatocellular carcinoma (HCC) is the sixth most common cancer man liver carboxylesterase 1; HP, haptoglobin; LCH, lentil lectin; pCD, procathepsin D; ROC, receiving operating characteristic ∗ Additional corresponding author: Professor Yuan-Fang Ma, E-mail: [email protected]

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and the third leading cause of cancer mortality worldwide [1]. At initial diagnosis, only 10–30% of HCC patients have surgically resectable disease and the 5-year survival rate is less than 5% [2]. The dismal prognosis is in part attributable to diagnosis at an advanced stage, when few effective therapies are available. Therefore, surveillance of high-risk subjects, such as cirrhotic subjects and HBV or HCV carriers, has the potential to reduce HCC-related mortality by detecting HCC at an early stage. Several groups have provided evidence that early detection of HCC through surveillance does result in reduced mortality [3]. Current approaches for HCC surveillance consist of ultrasound scanning and serum ␣-fetoprotein (AFP) estimation at 6-month intervals. However, ultrasound is of limited clinical value for screening due to operator dependency, a minimum tumor size of 3 cm for detection, and limited ability to differentiate malignant from benign lesions. On the other hand, other imaging methods such as triphasic computed tomography scan and magnetic resonance imaging with intravenous gadolinium are time-consuming and too expensive for routine screening [4]. Several serum biomarkers have been proposed for the diagnosis of HCC, either alone or in combination, which include AFP, ␣1–6 fucosylated AFP (AFP-L3), des-gamma carboxyprothrombin (DCP), Golgi protein 73 (GP73), isozyme of alkaline phosphatase specific for HCC (variant ALP), and isozyme of ␥-glutamyl transpeptidase specific for HCC (novel ␥-GTP) [5, 6]. The sensitivity and specificity of these markers limit their utility for HCC surveillance. For example, the reported sensitivity of 61 to 75% for AFP-L3 [7] and the specificity of 90% for DCP [8] were superior to others at the time of HCC diagnosis. However, high-serum AFP-L3 was closely associated with poor differentiation of HCC [9], and the sensitivity of DCP for early and small HCC ( 125). The areas under the ROC curves for cirrhotic controls versus noncirrhotic controls and HCC versus cirrhotic controls were 0.70 (95% CI: 0.42–0.98, sensitivity 83%, specificity 64%, cutoff > 36) and 0.79 (95% CI: 0.51–0.10, sensitivity 85%, specificity 67%, cutoff > 125), respectively (Fig. 5B–D).  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Discussion

Early diagnosis of HCC is the key to effective therapy as potentially curative treatments, such as surgical resection, radiofrequency ablation, and orthotopic liver transplantation, are only applicable to patients with tumors less than 5 cm in diameter. This observation has led to routine screening/surveillance of patients at high risk of HCC, namely those with advanced chronic liver disease and certain carriers of the Hepatitis B or C viruses. Among the serological tests proposed, alphafetoprotein is the most widely used, but its sensitivity and specificity are limited, especially in the early stage of the disease and thus alternative serum markers would be valuable [29]. With a view to identifying biomarker candidates for HCC diagnosis and potential therapeutic targets, we performed Nlinked glycoproteome profiling of human HCC and normal liver. A combination of three lectins, Con A, LCH, and GNA, was used to isolate N-linked glycoproteins. Subsequently, 28 proteins with a significant difference between HCC and nontumorous tissue were identified and we selected three glycoproteins, CD, hCE1, and HP, as HCC biomarker candidates for preliminary validation. Another implication of our study is that pCD expressed in HCC has a glycan structure that changes during the development and progression of HCC, as is the case for AFP [18], GP73 [19], HP [24, 25], and kallikrein 6 [30]. Development of a high-throughput method, such as a lectin-based ELISA for

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Figure 5. Serum levels of ConA-pCD and ROC curve for diagnosis of HCC versus non-HCC. (A) The box indicates that serum level of ConA-pCD in noncirrhotic controls (N = 26), cirrhotic controls (N = 29), and HCC (N = 55) was 60.8 ± 85.6, 140.8 ± 159.6, 368.4 ± 366.7, respectively, by signal intensity quantification. (B–D) The ROC curves show the diagnostic accuracy in optimal cut-off value (open triangle) to discriminate cirrhotic controls from noncirrhotic controls (B), HCCs from cirrhotic controls (C), and non-HCC controls (D). The area under the ROC curve for each comparison group was 0.70, 0.79, and 0.88, respectively.

glycan analysis, is currently being explored by our group, aiming to test the diagnostic value of serum level of ConA-pCD with larger number of HCC cases and controls. An increasing number of studies suggest overexpression and/or hypersecretion of CD/pCD in various types of cancer [31]. CD facilitates the development and progression of cancer in various phases of tumorigenesis [32, 33]. Depletion of CD or pCD expression in a highly metastatic MDA-MB231 human breast cancer cell line resulted in significant decreased in vitro and in vivo invasion and metastatic ability [34]. Impaired in vitro invasive ability of SNU449 and SNU473 was also demonstrated in our study after siRNA-mediated knockdown of CD/pCD. Consistent with these experimental data, a large number of clinical studies have reported that CD/pCD is an independent biomarker for poor prognosis in multiple types of malignancies and elevated expression of CD/pCD is associated with an increased risk of relapse and metastasis [32, 33]. These observations further support the potential of CD/pCD as a biomarker for HCC detection and characterization and as a target for therapeutic intervention.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Deregulation of the intracellular routing of CD causes increased pCD secretion into the extracellular milieu under pathological conditions, such as cancer [35]. Metastatic breast cancer cell lines secreted up to 50% of their pCD in vitro [36], whereas pCD accounted for only 10% of total CD in tumor cells and was undetectable in normal mammary epithelial cells by immunostaining [35]. Besides CD rerouting, overexpression of pCD/CD in cancer contributes to the hypersecretion of pCD and direct secretion of mature CD from pericellular lysosomes of metastatic cancer cells has also been observed [37]. The secreted pCD/CD exerts growth stimulatory action in an autocrine or paracrine fashion within a tumor microenvironment [38]. Several hypotheses have been put forward to explain the aberrant secretion of CD/pCD by cancer cells [39]. Studies have shown that the secreted pCD from breast cancer cells MCF7 is remarkably heterogeneous and had more acidic isoforms of pCD compared to normal mammary cells [35]. In human HCC tissues, purified CD displayed another five to six more acidic isoforms (pI range 4.4–5.3) compared to

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CD from normal liver, which expressed distinct isoforms (pI 5.6, 6.1, 6.7). Following endo-␤-N-acetylglucosaminidase H treatment, the acidic components of either CD from human hepatoma or pCD from MCF7 cells disappeared and reverted to identical patterns of normal counterparts [40], suggesting that structural changes of high mannose or hybrid N-linked oligosacchrides may contribute to the acidic components of CD/pCD. In-depth analysis showed that HCC tissue contains twice the amount of mannose-6-phosphate as that of normal liver CD [40], but no investigations have been done to identify the groups responsible for the more acidic pCD in breast cancer cells. The more acidic pCD isoforms may represent an impairment of pCD routing to lysosomes. In agreement with these findings, our study shows that pCD purified from pooled HCC serum by ConA affinity chromatography presents as a train of at least 4 protein spots with marked up-regulation in comparison to pooled non-HCC control on 2D Western blot images. Our findings suggest that the aberrant glycan structure attached to pCD may account for rerouting of pCD trafficking in HCC cells resulting in secretion into the blood during HCC development and evolution. Furthermore, the proportion of ConA-pCD in serum displayed clear clinical value with good sensitivity and specificity for diagnosis of HCC. Development of a high-throughput quantitative assay for these HCC-specific glycoforms of pCD would aid in determining diagnostic accuracy in a larger cohort of patients. In addition, we found that either total HP or ConA-binding HP from HCC decreased significantly in HCC tissues. Serum levels of HP are reportedly elevated in malignant disease irrespective of cancer type [24, 25, 41, 42]. Thus, the decreased tissue levels could be due to an increased rate of HP secretion. Aberrant glycosylation of HP has been correlated with tumor development and progression, including increased ␤-(1, 6)branched N-linked glycan structures in breast cancer [43] and increased triantennary glycan in the HP ␤ chain in HCC [24]. We also found that levels of hCE1, an enzyme predominantly expressed in the liver, which plays an important role in drug metabolism, were decreased in HCC tissue. In agreement with our findings, marked under-expression of hCE1 in HCC tissue has been recently reported using a combination of five types of lectins to enrich N-linked glycoproteins from HCC. Furthermore, elevation of hCE1 levels in HCC plasma was shown after enrichment by immunoprecipitation [15]. In summary, our proteomic analysis of N-linked glycoprotein enriched by lectin affinity chromatograpy has identified HP, hCE1, and CD/pCD as candidate biomarkers associated with HCC development and progression. In-depth analysis of CD showed that ConA-pCD is a promising, novel serological biomarker for diagnosing HCC. Development of a high-throughput assay, such as a ConA lectin-ELISA, will be needed, in the setting of a large-scale clinical study, to thoroughly validate serum ConA-pCD as a biomarker for the diagnosis and prognosis of HCC.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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This study was supported by Cancer Research UK and the National Natural Science Founding of China (No. 81072039). The authors have declared no conflict of interest.

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Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Mu...
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