Plasma Peptidome as a Source of Biomarkers for Diagnosis of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is the bile duct cancer which constitutes one of the important public health problem in Thailand with high mortality rate, especially in Opisthorchis viverrini (the risk factor of CCA) endemic area of the northeastern region of the country (Sripa and Pairojkul, 2008; Pinlaor et al., 2013). Most patients are present to the hospitals or health facilities at the later stage of disease progression due to asymptomatic and unavailability of diagnostic tool for early detection of the tumor. These factors together with limited effective therapeutic treatment options, have led to unsatisfactory control of this type of cancer. Identification of potential biomarkers for early diagnosis of CCA is required (Jia et al., 2014; Xu et al., 2014). Several attempts have been successfully identified the potential peptidome biomarkers for other types of cancer as well as infectious diseases (Terracciano et al., 2011; Xiao et al., 2011; Fan et al., 2012; Yang et al., 2012). The peptidome includes small proteins (10 kDa or smaller), cleaved proteins, and protease digested proteins. Both the pattern and the level of these expressed peptides in disease conditions can be exploited as biomarkers for disease diagnosis (Fukuoka


Introduction
Cholangiocarcinoma (CCA) is the bile duct cancer which constitutes one of the important public health problem in Thailand with high mortality rate, especially in Opisthorchis viverrini (the risk factor of CCA) endemic area of the northeastern region of the country (Sripa and Pairojkul, 2008;Pinlaor et al., 2013).Most patients are present to the hospitals or health facilities at the later stage of disease progression due to asymptomatic and unavailability of diagnostic tool for early detection of the tumor.These factors together with limited effective therapeutic treatment options, have led to unsatisfactory control of this type of cancer.Identification of potential biomarkers for early diagnosis of CCA is required (Jia et al., 2014;Xu et al., 2014).Several attempts have been successfully identified the potential peptidome biomarkers for other types of cancer as well as infectious diseases (Terracciano et al., 2011;Xiao et al., 2011;Fan et al., 2012;Yang et al., 2012).The peptidome includes small proteins (10 kDa or smaller), cleaved proteins, and protease digested proteins.Both the pattern and the level of these expressed peptides in disease conditions can be exploited as biomarkers for disease diagnosis (Fukuoka

Samples
Plasma samples obtained from (i) 20 CCA patients (intrahepatic cholangiocarcinoma, 13 males and 7 females, aged 36-72 years), (ii) 12 patients with Opisthorchis viverrini (OV, 9 males and 3 females, aged 32-71 years) infection, and and 8 healthy subjects (7 males and 1 female, aged 40-63 years) were kindly provided by Dr. Petcharin Srivatanakul, the National Cancer Institute of Thailand.Ethical approval of the study protocol was obtained from the Ethics Committee of the Ministry of Public Health of Thailand.Written informed consent for study participation was obtained from all subjects prior to the study.The diagnosis of CCA was based on clinical and histopathological examination.The levels of liver enzymes (AFP, AST, ALT, and ALP) and CA19-9 in CCA patients were significantly higher than non-CCA subjects in group (ii) and (iii) [median (range): AFP 3.17 (1.47-291.7) vs. 3.47 (1.59-9.2) vs. 2.77 (1.23-4.39)U/ml; AST 68 ) U/ml for group (i) vs. (ii) vs. (iii), respectively, Mann-Whitney U test, p < 0.05].Plasma peptide concentrations were determined using Lowry's method (Lowry et al., 1951) with bovine serum albumin (SIGMA-Aldrich, MO, USA) as a reference standard.

Preparation of plasma peptides and LC-MS/MS analysis
Plasma peptides (≤ 10 kDa in size) from CCA patients and non-CCA subjects were isolated using a 10 kDa cutoff column (Pall corporation, NY, USA).Plasma sample (200 µl) was loaded into and separated from the column through centrifugation at 7,000 ×g for 30 minutes.The separated plasma peptides were concentrated using vacuum concentrator and treated with buffer I (10 mM each of dithiothreitol and ammonium bicarbonate) and buffer II (100 mM iodoacetamide and 10 mM ammonium bicarbonate) for carbamidomethyl reaction at room temperature.The reaction was terminated by the addition of buffer I. Finally, plasma peptides were digested with trypsin (10 ng trypsin in 50% acetonitrile and 10 mM ammonium bicarbonate) through an overnight incubation at room temperature and samples were injected into LC-MS/MS.

Analysis of LC-MS/MS data
The LC-MS/MS data were analyzed using DeCyder MSTM (Amersham Bioscience AB, Uppsala, Sweden) and MASCOT (http://www.matrixscience.com)programs.Gene ontology and signaling pathways were analyzed using STRAP (Vivek Bhatia, Boston University School of Medicine) and PANTHER (http://www.pantherdb.org)(Mi et al., 2013), respectively.The metabolic, regulatory, and biosynthesis of secondary metabolites pathways were analyzed using iPath (http://pathways.embl.de/iPath2.cgi)(Yamada et al., 2011).The presence of specific peptides in either the CCA patients or non-CCA subjects were investigated to identify potential biomarkers for early diagnosis of CCA.The protein-protein interactions of potential biomarkers were investigated using STRING (http://string-db.org/) to elucidate the involvement of potential biomarkers in carcinogenesis.

Results
Analysis of CCA plasma peptidome A total of 209 peptides were obtained from all plasma samples (CCA patients and non-CCA subjects) and identified from LC-MS/MS data by DeCyder MSTM and MASCOT programs.Out of these, 148 and 115 peptides were retrieved from UniProt ID and KEGG, respectively.The UniProt ID retrieved peptides were further analyzed by STRAP for identification of their functions.Results suggest that most of these peptides were involved in cellular process in biological function category, nucleus in cellular component category, and binding in molecular function category (Figure 1A,1B,and 1C,respectively).Analysis of signaling pathway related peptides by PANTHER showed 15 peptides having a role in cell signaling such as growth factor receptor-, hormone receptor-, cadherin-, and integrin-signaling pathways (Table 1).iPath analysis suggested the involvement of 12 peptides in metabolic pathway, e.g., cytosolic purine 5'-nucleotidase of purine metabolism, chain A, structure of human placental S-adenosylhomocysteine hydrolase (P23526) of cysteine and methionine metabolism, and peroxisomal sarcosine oxidase (Q9P0Z9) of amino acid metabolism.In addition, some are also involved in regulation and biosynthesis of secondary metabolite pathways (Table 2).

Identification of potential biomarkers for CCA from plasma peptidome
Out of the 209 peptides, three were specifically detected in plasma samples of the CCA patients but not in non-CCA subjects.These included ETS domain-  No.P27986) (Figure 2).

Discussion
Analysis of plasma peptidome of CCA patients identified only 5 out of 209 peptides as potential biomarkers for CCA, i.e., ETS domain-containing transcription factor ERF, PIK3CB, casein kinase II subunit alpha, KIAA0220, and LP2209.The first three have been reported to play major roles in signaling pathways that are associated with cell growth and proliferation (Zaldumbide et al., 2002;Jia et al., 2008).ETS domain-containing transcription factor ERF is a transcription repressor of ETS2 promoter which is regulated by MAPK1/ERK2 phosphorylation (Sgouras et al., 1995).PIK3CB plays a role in phosphatidylinositol (PI) signaling pathway (Wee et al., 2008;Dbouk et al., 2010) through interacting with AKT1, PTEN, and PIK3R1 (Figure 2).Up-regulation of this protein may affect the downstream signaling cascade of PI signaling pathway (Wee et al., 2008), which may promote cancer cell growth and differentiation.Casein kinase (CK) II subunit alpha is involved in cell proliferation.Inhibition of CK II kinase was demonstrated to activate cell apoptosis in cancer cell (Hamacher et al., 2007).In addition, the protein was shown to be activated following Wnt signaling pathway activation via frizzled receptor (Song et al., 2000;Seldin et al., 2005).The functions of KIAA0220 and LP2209 remain unclear.KIAA0220 cDNA has been characterized as nuclear pore complex-interacting protein, while LP2209 has been shown to promote mice NIH/3T3 cells' growth.The combination of these identified biomarkers could be applied as a battery of test for CCA diagnosis for diagnosis of CCA alone or CCA with OV infection (Table 4).The efficiency of the test for CCA diagnosis should be confirmed in a larger number of patients.

Figure
Figure 1.STRAP analysis of the functions of identified proteins based on (A) biological process, (B) cellular component, and (C) molecular function