Identification of potential pancreatic cancer serum markers: Increased sialyl-Lewis X on ceruloplasmin

Highlights

• Glycoproteomic strategy to find cancer markers based on changes in sLe^x antigen.

• Identification of proteins with increased sLe^x epitope in pancreatic cancer by MS.

• Ceruloplasmin N-glycan analysis confirmed an increase in sLe^x in pancreatic cancer.

• Ratio sLe^x on ceruloplasmin/ceruloplasmin is increased in most pancreatic cancer sera.

Abstract

Pancreatic adenocarcinoma (PDAC) usually shows an enhanced expression of sialyl-Lewis X (sLe^x) and related epitopes. PDAC may secrete some of the proteins carrying such increased sLe^x determinant into serum, so they could be used as PDAC markers. Previously, we identified acute-phase proteins with increased sLe^x in both PDAC and in chronic pancreatitis patients. In this study, depleted sera from the main acute-phase proteins has been analysed for the search of proteins with increased sLe^x levels in PDAC. Sera from healthy controls, chronic pancreatitis and PDAC patients were depleted, electrophoresed and subjected to sLe^x immunodetection. Proteins that differentially expressed sLe^x in PDAC were trypsin digested and identified by LC-ESI-QTOF mass spectrometry. Five protein bands that differentially expressed sLe^x in PDAC were identified and corresponded to seven different acute-phase proteins. Among them, ceruloplasmin (CP) was selected for further analysis. N-glycan sequencing of CP confirmed the increase of sLe^x levels in CP in PDAC patients. Healthy controls, chronic pancreatitis and PDAC patients' sera were immunoprecipitated with anti-CP antibodies, and their sLe^x and CP levels were analysed by western blot. The sLe^x/CP ratio tended to be higher for the PDAC group, which altogether suggests that the sLe^x/CP ratio could be a useful biomarker for PDAC.

Introduction

Pancreatic cancer (PDAC) has the lowest 5-year survival rate (about 5%) of all cancer types. Although only representing around 3% of all cancer cases, it was the fourth leading cause of cancer death in Europe and the United States [1]. This poor survival may be attributed to its late diagnosis, usually performed after metastases have occurred. Early detection of pancreatic cancer would improve 5-year survival rate to 20% [1], [2].

CA19-9 serum detection is currently used to monitor PDAC patients. However, its use in diagnosis is restricted by its false positive results, as it is also increased in patients with benign pancreaticobiliary disorders such as chronic pancreatitis (ChrP) [3], [4]. Thus, the availability of adequate biomarkers for PDAC detection is of major interest.

Glycosylation changes are a universal feature of malignant transformation and tumour progression. These changes can be found either in tumour cell surface or in secreted glycoconjugates. Glycan changes in malignant cells take a variety of forms, usually affecting terminal glycan structures [5]. In particular, sialyl-Lewis X (sLe^x) and related Lewis antigens have been found to be overexpressed in PDAC cell lines [6], [7] and tissues [8], [9], [10]. An increase of sialylated Lewis antigens and both fucosylation and sialylation of certain glycoproteins have been detected in the sera of PDAC patients compared to healthy individuals and ChrP patients [11], [12], [13]. These data suggest that pancreatic tumour may shed into the blood glycoproteins carrying sLe^x, which could be used as PDAC tumour markers.

In a previous work, we identified serum glycoproteins carrying increased sLe^x in both advanced PDAC and chronic pancreatitis patients [14]. However, these proteins corresponded to major acute-phase proteins (APP); alpha-1-acid-glycoprotein, haptoglobin and transferrin, which are produced mainly by the liver. Other APPs were also found to bear increased sLe^x levels only in chronic pancreatitis patients (alpha-1-antitrypsin and fetuin). Although sLe^x on these APPs may be used as cancer prognostic factors, these modifications are not specific enough to be used as PDAC markers.

In the present work, a glycoproteomic strategy to identify potential pancreatic cancer biomarkers based on changes in sLe^x glycan antigen in serum proteins was performed. For this purpose, the most abundant serum proteins were depleted in order to identify other glycoproteins with enhanced sLe^x from PDAC patients and have found ceruloplasmin (CP) as an interesting candidate for further analysis.

CP is an acute-phase protein produced by the liver and secreted in plasma. Its function is related to copper transport in serum and it is suggested to have a role in cancer since it is involved in angiogenesis and neovascularisation [15], [16]. CP has 4 described N-glycosylation sites with complex type, bi, tri and tetrantennary structures both sialylated and fucosylated, containing sLe^x epitope mainly in triantennary structures, but also bi- and tetra-antennary [17], [18]. In this study, the sLe^x levels on CP from sera of PDAC, ChrP and healthy controls were analysed and tended to be increased in the PDAC group.