Plasma Phosphoproteome and Differential Plasma Phosphoproteins with Opisthorchis Viverrini-Related Cholangiocarcinoma

  • Kotawong, Kanawut (Graduate Program in Bioclinical Sciences, Thammasat University) ;
  • Thitapakorn, Veerachai (Graduate Program in Bioclinical Sciences, Thammasat University) ;
  • Roytrakul, Sittiruk (Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency) ;
  • Phaonakrop, Narumon (Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency) ;
  • Viyanant, Vithoon (Graduate Program in Bioclinical Sciences, Thammasat University) ;
  • Na-Bangchang, Kesara (Graduate Program in Bioclinical Sciences, Thammasat University)
  • Published : 2015.03.04


This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.


Supported by : National Research University (NRU)


  1. Bai Y, Luo Y, Liu S, et al (2011). PRL-1 protein promotes ERK1/2 and RhoA protein activation through a noncanonical interaction with the Src homology 3 domain of p115 Rho GTPase-activating protein. J Biol Chem, 286, 42316-24.
  2. Bian S, Sun X, Bai A, et al (2013). P2X7 integrates PI3K/AKT and AMPK-PRAS40-mTOR signaling pathways to mediate tumor cell death. PLoS One, 8, 60184.
  3. Burak K, Angulo P, Pasha TM, et al (2004). Incidence and risk factors for cholangiocarcinoma in primary sclerosing cholangitis. Am J Gastroenterol, 99, 523-6.
  4. Deng BG, Yao JH, Liu QY, et al (2013). Comparative serum proteomic analysis of serum diagnosis proteins of colorectal cancer based on magnetic bead separation and Maldi-tof mass spectrometry. Asian Pac J Cancer Prev, 14, 6069-75.
  5. Eaton JE, Talwalkar JA, Lazaridis KN, et al (2013). Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management. Gastroenterology, 145, 521-36.
  6. Eichhorn PJ, Creyghton MP, Bernards R (2009). Protein phosphatase 2A regulatory subunits and cancer. Biochim Biophys Acta, 1795, 1-15.
  7. Ge F, Xiao CL, Yin XF, et al (2010). Phosphoproteomic analysis of primary human multiple myeloma cells. J Proteomics, 73, 1381-90.
  8. Goni BW, Yusuph H, Mustapha SK, et al (2013). Hepatic transaminase and alkaline phosphatase enzyme levels in HIV/HBV co-infected and HIV mono-infected patients in Maiduguri, Nigeria. Niger J Clin Pract, 16, 530-4.
  9. Guha U, Chaerkady R, Marimuthu A, et al (2008). Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci USA, 105, 14112-7.
  10. Kang YK, Kim WH, Jang JJ (2002). Expression of G1-S modulators (p53, p16, p27, cyclin D1, Rb) and Smad4/Dpc4 in intrahepatic cholangiocarcinoma. Hum Pathol, 33, 877-83.
  11. Khoontawad J, Hongsrichan N, Chamgramol Y, et al (2014). Increase of exostosin 1 in plasma as a potential biomarker for opisthorchiasis-associated cholangiocarcinoma. Tumour Biol, 35, 1029-39.
  12. Kunutsor SK, Apekey TA, Walley J (2013). Liver aminotransferases and risk of incident type 2 diabetes: a systematic review and meta-analysis. Am J Epidemiol, 178, 159-71.
  13. Lee D, Do IG, Choi K, et al (2012). The expression of phospho- AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas. Mod Pathol, 25, 131-9.
  14. Letourneux C, Rocher G, Porteu F (2006). B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK. EMBO J, 25, 727-38.
  15. Li P, Yang J, Ma QY, et al (2013). Biomarkers screening between preoperative and postoperative patients in pancreatic cancer. Asian Pac J Cancer Prev, 14, 4161-5.
  16. Liang D, Zeng Q, Xu Z, et al (2014). BAFF activates Erk1/2 promoting cell proliferation and survival by Ca2+-CaMKII dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells. Biochem Pharmacol, 87, 332-43.
  17. Liao MH, Xiang YC, Huang JY, et al (2013). The disturbance of hippocampal CaMKII/PKA/PKC phosphorylation in early experimental diabetes mellitus. CNS Neurosci Ther, 19, 329-36.
  18. Lien SC, Chang SF, Lee PL, et al (2013). Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK. Biochim Biophys Acta, 1833, 3124-33.
  19. Lin J, Adam RM, Santiestevan E, et al (1999). The phosphatidylinositol 3'-kinase pathway is a dominant growth factor-activated cell survival pathway in LNCaP human prostate carcinoma cells. Cancer Res, 59, 2891-7.
  20. Lowry OH, Rosebrough NJ, Farr AL, et al (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193, 265-75.
  21. Ma L, Chen Z, Erdjument-Bromage H, et al (2005). Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell, 121, 179-93.
  22. Mackinnon A (2000). A spreadsheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement. Comput Biol Med, 30, 127-34.
  23. Malaguarnera G, Paladina I, Giordano M, et al (2013). Serum markers of intrahepatic cholangiocarcinoma. Dis Markers, 34, 219-28.
  24. Martins-de-Souza D, Guest PC, Vanattou-Saifoudine N, et al (2012). Phosphoproteomic differences in major depressive disorder postmortem brains indicate effects on synaptic function. Eur Arch Psychiatry Clin Neurosci, 262, 657-66.
  25. Mi H, Muruganujan A, Casagrande JT, et al (2013). Large-scale gene function analysis with the PANTHER classification system. Nat Protoc, 8, 1551-66.
  26. Mitacek EJ, Brunnemann KD, Suttajit M, et al (1999). Exposure to N-nitroso compounds in a population of high liver cancer regions in Thailand: volatile nitrosamine (VNA) levels in Thai food. Food Chem Toxicol, 37, 297-305.
  27. Moolthiya P, Tohtong R, Keeratichamroen S, et al (2014). Role of mTOR inhibitor in cholangiocarcinoma cell progression. Oncol Lett, 7, 854-60.
  28. Rodgers JT, Vogel RO, Puigserver P (2011). Clk2 and B56beta mediate insulin-regulated assembly of the PP2A phosphatase holoenzyme complex on Akt. Mol Cell, 41, 471-9.
  29. Sacher M, Jiang Y, Barrowman J, et al (1998). TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J, 17, 2494-503.
  30. Sahani D, Prasad SR, Tannabe KK, et al (2003). Thorotrastinduced cholangiocarcinoma: case report. Abdom Imaging, 28, 72-4.
  31. Skeen JE, Bhaskar PT, Chen CC, et al (2006). Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner. Cancer Cell, 10, 269-80.
  32. Sripa B, Kaewkes S, Sithithaworn P, et al (2007). Liver fluke induces cholangiocarcinoma. PLoS Med, 4, 201.
  33. Srisomsap C, Sawangareetrakul P, Subhasitanont P, et al (2010). Proteomic studies of cholangiocarcinoma and hepatocellular carcinoma cell secretomes. J Biomed Biotechnol, 2010, 437143.
  34. Sriwanitchrak P, Viyanant V, Chaijaroenkul W, et al (2011). Proteomics analysis and evaluation of biomarkers for detection of cholangiocarcinoma. Asian Pac J Cancer Prev, 12, 1503-10.
  35. Thamavit W, Bhamarapravati N, Sahaphong S, et al (1978). Effects of dimethylnitrosamine on induction of cholangiocarcinoma in Opisthorchis viverrini-infected Syrian golden hamsters. Cancer Res, 38, 4634-9.
  36. Tyson GL, El-Serag HB (2011). Risk factors for cholangiocarcinoma. Hepatology, 54, 173-84.
  37. van Abel D, Abdulhamid O, Scheper W, et al (2012). STOX1A induces phosphorylation of tau proteins at epitopes hyperphosphorylated in Alzheimer's disease. Neurosci Lett, 528, 104-9.
  38. van Abel D, Abdulhamid O, Scheper W, et al (2012). STOX1A induces phosphorylation of tau proteins at epitopes hyperphosphorylated in Alzheimer's disease. Neurosci Lett, 528, 104-9.
  39. Wang X, Stewart PA, Cao Q, et al (2011). Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry. J Proteome Res, 10, 3920-8.
  40. White NM, Masui O, Desouza LV, et al (2014). Quantitative proteomic analysis reveals potential diagnostic markers and pathways involved in pathogenesis of renal cell carcinoma. Oncotarget.
  41. Yamada T, Letunic I, Okuda S, et al (2011). iPath2.0: interactive pathway explorer. Nucleic Acids Res, 39, 412-5.
  42. Yang CY, Chang CH, Yu YL, et al (2008). PhosphoPOINT: a comprehensive human kinase interactome and phosphoprotein database. Bioinformatics, 24, 14-20.
  43. Yonglitthipagon P, Pairojkul C, Chamgramol Y, et al (2012). Prognostic significance of peroxiredoxin 1 and ezrin-radixinmoesin- binding phosphoprotein 50 in cholangiocarcinoma. Hum Pathol, 43, 1719-30.
  44. Zhang S, Chen Y, Zhu Z, et al (2013). Differential expression of carbohydrate antigen 19-9 in human colorectal cancer: A comparison with colon and rectal cancers. Mol Clin Oncol, 1, 1072-8.