Tumor Biology

, Volume 35, Issue 10, pp 10031–10041 | Cite as

Increased EphB2 expression predicts cholangiocarcinoma metastasis

  • Walaiporn Khansaard
  • Anchalee Techasen
  • Nisana Namwat
  • Puangrat Yongvanit
  • Narong Khuntikeo
  • Anucha Puapairoj
  • Watcharin Loilome
Research Article


The activation of Ephrin (Eph) receptors, the largest tyrosine kinase families of cell surface receptor, has recently been addressed in human cholangiocarcinoma (CCA). Therefore, the present study aimed to investigate the role of Eph receptors and its ligands in CCA. Of all 50 cases of human CCA tested, immunohistochemical staining demonstrated that EphB2, EphB4, ephrinB1, and ephrinB2 were 100 % positive in CCA tissues with overexpressions of the above proteins as 56, 56, 70, and 48 % of cases, respectively. High expression of EphB2 was significantly correlated with the metastatic status of patients (P = 0.027). We also found that the high co-expression level of EphB2/ephrinB1 or EphB2/ephrinB2 were significantly correlated with the metastatic status of the patients (P = 0.034 and P = 0.024). Furthermore, we showed that the high co-expression level of EphB4/MVD and ephrinB1/MVD were significantly correlated with the metastasis status of CCA patients (P = 0.012 and P = 0.029). We further demonstrated that the EphB2 suppression using siRNA significantly reduced CCA cell migration by decreasing the phosphorylation of focal adhesion kinase (FAK) and paxillin. In conclusion, the upregulation of EphB2 receptors and its specific ligands (ephrinB1 and ephrinB2) leads to CCA metastasis. Suppression of EphB2 expression as well as inhibition of its downstream signaling proteins might serve as possible therapeutic strategies in human CCA.


EphB2 Tumor progression Metastasis Cholangiocarcinoma 



This study is funded by the grant from the Thailand Research Fund (Grant No. MRG5400834) and the Research Assistantship Grant of the Faculty of Medicine, Khon Kaen University (Grant No. AS55203) to WL along with the grants from the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster (SHeP-GMS), Khon Kaen University allocated to WK and WL. We thank Prof. Ross H. Andrews for English language assistance.

Conflicts of interest


Supplementary material

13277_2014_2295_MOESM1_ESM.docx (38 kb)
Supplementary Table 1 (DOCX 37 kb)
13277_2014_2295_MOESM2_ESM.docx (38 kb)
Supplementary Table 2 (DOCX 38 kb)
13277_2014_2295_MOESM3_ESM.docx (36 kb)
Supplementary Table 3 (DOCX 35 kb)


  1. 1.
    Vatanasapt V, Sriamporn S, Vatanasapt P. Cancer control in Thailand. Jpn J Clin Oncol. 2002;32(Suppl):S82–91.CrossRefPubMedGoogle Scholar
  2. 2.
    Dechakhamphu S, Pinlaor S, Sitthithaworn P, Bartsch H, Yongvanit P. Accumulation of miscoding etheno-DNA adducts and highly expressed DNA repair during liver fluke-induced cholangiocarcinogenesis in hamsters. Mutat Res. 2010;691:9–16.CrossRefPubMedGoogle Scholar
  3. 3.
    Dechakhamphu S, Pinlaor S, Sitthithaworn P, Nair J, Bartsch H, Yongvanit P. Lipid peroxidation and etheno DNA adducts in white blood cells of liver fluke-infected patients: protection by plasma alpha-tocopherol and praziquantel. Cancer Epidemiol Biomarkers Prev. 2010;19:310–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Dechakhamphu S, Yongvanit P, Nair J, Pinlaor S, Sitthithaworn P, Bartsch H. High excretion of etheno adducts in liver fluke-infected patients: protection by praziquantel against DNA damage. Cancer Epidemiol Biomarkers Prev. 2008;17:1658–64.CrossRefPubMedGoogle Scholar
  5. 5.
    Pinlaor S, Yongvanit P, Hiraku Y, Ma N, Semba R, Oikawa S, et al. 8-nitroguanine formation in the liver of hamsters infected with opisthorchis viverrini. Biochem Biophys Res Commun. 2003;309:567–71.CrossRefPubMedGoogle Scholar
  6. 6.
    Thamavit W, Bhamarapravati N, Sahaphong S, Vajrasthira S, Angsubhakorn S. Effects of dimethylnitrosamine on induction of cholangiocarcinoma in opisthorchis viverrini-infected syrian golden hamsters. Cancer Res. 1978;38:4634–9.PubMedGoogle Scholar
  7. 7.
    Thanan R, Murata M, Pinlaor S, Sithithaworn P, Khuntikeo N, Tangkanakul W, et al. Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine in patients with parasite infection and effect of antiparasitic drug in relation to cholangiocarcinogenesis. Cancer Epidemiol Biomarkers Prev. 2008;17:518–24.CrossRefPubMedGoogle Scholar
  8. 8.
    Loilome W, Juntana S, Namwat N, Bhudhisawasdi V, Puapairoj A, Sripa B, et al. Prkar1a is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma. Int J Cancer. 2011;129:34–44.CrossRefPubMedGoogle Scholar
  9. 9.
    Loilome W, Yongvanit P, Wongkham C, Tepsiri N, Sripa B, Sithithaworn P, et al. Altered gene expression in opisthorchis viverrini-associated cholangiocarcinoma in hamster model. Mol Carcinog. 2006;45:279–87.CrossRefPubMedGoogle Scholar
  10. 10.
    Techasen A, Loilome W, Namwat N, Takahashi E, Sugihara E, Puapairoj A, et al. Myristoylated alanine-rich c kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase c-dependent pathway. Cancer Sci. 2010;101:658–65.CrossRefPubMedGoogle Scholar
  11. 11.
    Dokduang H, Juntana S, Techasen A, Namwat N, Yongvanit P, Khuntikeo N, et al. Survey of activated kinase proteins reveals potential targets for cholangiocarcinoma treatment. Tumour Biol. 2013;34:3519–28.CrossRefPubMedGoogle Scholar
  12. 12.
    Loilome W, Bungkanjana P, Techasen A, Namwat N, Yongvanit P, Puapairoj A, et al. Activated macrophages promote Wnt/beta-catenin signaling in cholangiocarcinoma cells. Tumour Biol. 2014;35:5357–67.CrossRefPubMedGoogle Scholar
  13. 13.
    Yothaisong S, Dokduang H, Techasen A, Namwat N, Yongvanit P, Bhudhisawasdi V, et al. Increased activation of PI3K/AKT signaling pathway is associated with cholangiocarcinoma metastasis and PI3K/mTOR inhibition presents a possible therapeutic strategy. Tumour Biol. 2013;34:3637–48.CrossRefPubMedGoogle Scholar
  14. 14.
    Mosch B, Reissenweber B, Neuber C, Pietzsch J. Eph receptors and ephrin ligands: important players in angiogenesis and tumor angiogenesis. J Oncol. 2010;2010:135285.PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008;133:38–52.CrossRefPubMedGoogle Scholar
  16. 16.
    Pasquale EB. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer. 2010;10:165–80.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Surawska H, Ma PC, Salgia R. The role of ephrins and eph receptors in cancer. Cytokine Growth Factor Rev. 2004;15:419–33.CrossRefPubMedGoogle Scholar
  18. 18.
    Nakada M, Niska JA, Miyamori H, McDonough WS, Wu J, Sato H, et al. The phosphorylation of EphB2 receptor regulates migration and invasion of human glioma cells. Cancer Res. 2004;64:3179–85.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang SD, Rath P, Lal B, Richard JP, Li Y, Goodwin CR, et al. EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase. Oncogene. 2012;31:5132–43.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Sikkema AH, den Dunnen WF, Hulleman E, van Vuurden DG, Garcia-Manero G, Yang H, et al. EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion. Neuro Oncol. 2012;14:1125–35.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Kinch MS, Moore MB, Harpole Jr DH. Predictive value of the EphA2 receptor tyrosine kinase in lung cancer recurrence and survival. Clin Cancer Res. 2003;9:613–8.PubMedGoogle Scholar
  22. 22.
    Lawrenson ID, Wimmer-Kleikamp SH, Lock P, Schoenwaelder SM, Down M, Boyd AW, et al. Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling. J Cell Sci. 2002;115:1059–72.PubMedGoogle Scholar
  23. 23.
    Liu W, Jung YD, Ahmad SA, McCarty MF, Stoeltzing O, Reinmuth N, et al. Effects of overexpression of ephrin-B2 on tumour growth in human colorectal cancer. Br J Cancer. 2004;90:1620–6.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Macrae M, Neve RM, Rodriguez-Viciana P, Haqq C, Yeh J, Chen C, et al. A conditional feedback loop regulates ras activity through EphA2. Cancer Cell. 2005;8:111–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Adams RH, Wilkinson GA, Weiss C, Diella F, Gale NW, Deutsch U, et al. Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 1999;13:295–306.PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Brantley-Sieders DM, Zhuang G, Hicks D, Fang WB, Hwang Y, Cates JM, et al. The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling. J Clin Invest. 2008;118:64–78.PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med. 2007;17:145–51.CrossRefPubMedGoogle Scholar
  28. 28.
    Sawamiphak S, Seidel S, Essmann CL, Wilkinson GA, Pitulescu ME, Acker T, et al. Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature. 2010;465:487–91.CrossRefPubMedGoogle Scholar
  29. 29.
    Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998;93:741–53.CrossRefPubMedGoogle Scholar
  30. 30.
    Chiu ST, Chang KJ, Ting CH, Shen HC, Li H, Hsieh FJ. Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells. Carcinogenesis. 2009;30:1475–86.CrossRefPubMedGoogle Scholar
  31. 31.
    Oricchio E, Nanjangud G, Wolfe AL, Schatz JH, Mavrakis KJ, Jiang M, et al. The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma. Cell. 2011;147:554–64.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Jamnongkan W, Techasen A, Thanan R, Duenngai K, Sithithaworn P, Mairiang E, et al. Oxidized alpha-1 antitrypsin as a predictive risk marker of opisthorchiasis-associated cholangiocarcinoma. Tumour Biol. 2013;34:695–704.CrossRefPubMedGoogle Scholar
  33. 33.
    Namwat N, Puetkasichonpasutha J, Loilome W, Yongvanit P, Techasen A, Puapairoj A, et al. Downregulation of reversion-inducing-cysteine-rich protein with kazal motifs (RECK) is associated with enhanced expression of matrix metalloproteinases and cholangiocarcinoma metastases. J Gastroenterol. 2011;46:664–75.CrossRefPubMedGoogle Scholar
  34. 34.
    Kimura H, Nakajima T, Kagawa K, Deguchi T, Kakusui M, Katagishi T, et al. Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry. Liver. 1998;18:14–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Nanashima A, Shibata K, Nakayama T, Tobinaga S, Araki M, Kunizaki M, et al. Relationship between microvessel count and postoperative survival in patients with intrahepatic cholangiocarcinoma. Ann Surg Oncol. 2009;16:2123–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Jones G, Jurkiewicz MJ, Bostwick J, Wood R, Bried JT, Culbertson J, et al. Management of the infected median sternotomy wound with muscle flaps. The emory 20-year experience. Ann Surg. 1997;225:766–76. discussion 776-768.PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Lackmann M, Boyd AW. Eph, a protein family coming of age: more confusion, insight, or complexity? Sci Signal. 2008;1:re2.CrossRefPubMedGoogle Scholar
  38. 38.
    Merlos-Suarez A, Batlle E. Eph-ephrin signalling in adult tissues and cancer. Curr Opin Cell Biol. 2008;20:194–200.CrossRefPubMedGoogle Scholar
  39. 39.
    Nievergall E, Lackmann M, Janes PW. Eph-dependent cell-cell adhesion and segregation in development and cancer. Cell Mol Life Sci. 2012;69:1813–42.CrossRefPubMedGoogle Scholar
  40. 40.
    Miao H, Burnett E, Kinch M, Simon E, Wang B. Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol. 2000;2:62–9.CrossRefPubMedGoogle Scholar
  41. 41.
    Parrinello S, Napoli I, Ribeiro S, Wingfield Digby P, Fedorova M, Parkinson DB, et al. EphB signaling directs peripheral nerve regeneration through Sox2-dependent schwann cell sorting. Cell. 2010;143:145–55.CrossRefPubMedGoogle Scholar
  42. 42.
    Yokote H, Fujita K, Jing X, Sawada T, Liang S, Yao L, et al. Trans-activation of EphA4 and FGF receptors mediated by direct interactions between their cytoplasmic domains. Proc Natl Acad Sci U S A. 2005;102:18866–71.PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Tanaka M, Ohashi R, Nakamura R, Shinmura K, Kamo T, Sakai R, et al. Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2. EMBO J. 2004;23:1075–88.PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Yang NY, Pasquale EB, Owen LB, Ethell IM. The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization. J Biol Chem. 2006;281:32574–86.CrossRefPubMedGoogle Scholar
  45. 45.
    Iida H, Honda M, Kawai HF, Yamashita T, Shirota Y, Wang BC, et al. EphrinA1 expression contributes to the malignant characteristics of {alpha}-fetoprotein producing hepatocellular carcinoma. Gut. 2005;54:843–51.PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Li X, Wang Y, Zhen H, Yang H, Fei Z, Zhang J, et al. Expression of EphA2 in human astrocytic tumors: correlation with pathologic grade, proliferation and apoptosis. Tumour Biol. 2007;28:165–72.CrossRefPubMedGoogle Scholar
  47. 47.
    Vaught D, Brantley-Sieders DM, Chen J. Eph receptors in breast cancer: roles in tumor promotion and tumor suppression. Breast Cancer Res. 2008;10:217.PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Gerety SS, Anderson DJ. Cardiovascular ephrinB2 function is essential for embryonic angiogenesis. Development. 2002;129:1397–410.PubMedGoogle Scholar
  49. 49.
    Gerety SS, Wang HU, Chen ZF, Anderson DJ. Symmetrical mutant phenotypes of the receptor ephB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell. 1999;4:403–14.CrossRefPubMedGoogle Scholar
  50. 50.
    Abengozar MA, de Frutos S, Ferreiro S, Soriano J, Perez-Martinez M, Olmeda D, et al. Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth. Blood. 2012;119:4565–76.CrossRefPubMedGoogle Scholar
  51. 51.
    Foubert P, Silvestre JS, Souttou B, Barateau V, Martin C, Ebrahimian TG, et al. Psgl-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells. J Clin Invest. 2007;117:1527–37.PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Mansson-Broberg A, Siddiqui AJ, Genander M, Grinnemo KH, Hao X, Andersson AB, et al. Modulation of ephrinB2 leads to increased angiogenesis in ischemic myocardium and endothelial cell proliferation. Biochem Biophys Res Commun. 2008;373:355–9.CrossRefPubMedGoogle Scholar
  53. 53.
    Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature. 1989;339:58–61.CrossRefPubMedGoogle Scholar
  54. 54.
    Tomisaki S, Ohno S, Ichiyoshi Y, Kuwano H, Maehara Y, Sugimachi K. Microvessel quantification and its possible relation with liver metastasis in colorectal cancer. Cancer. 1996;77:1722–8.CrossRefPubMedGoogle Scholar
  55. 55.
    Xi HQ, Wu XS, Wei B, Chen L. Aberrant expression of EphA3 in gastric carcinoma: correlation with tumor angiogenesis and survival. J Gastroenterol. 2012;47:785–94.CrossRefPubMedGoogle Scholar
  56. 56.
    Zatterstrom UK, Brun E, Willen R, Kjellen E, Wennerberg J. Tumor angiogenesis and prognosis in squamous cell carcinoma of the head and neck. Head Neck. 1995;17:312–8.CrossRefPubMedGoogle Scholar
  57. 57.
    Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005;6:56–68.CrossRefPubMedGoogle Scholar
  58. 58.
    Schaller MD. Paxillin: a focal adhesion-associated adaptor protein. Oncogene. 2001;20:6459–72.CrossRefPubMedGoogle Scholar
  59. 59.
    Guo DL, Zhang J, Yuen ST, Tsui WY, Chan AS, Ho C, et al. Reduced expression of EphB2 that parallels invasion and metastasis in colorectal tumours. Carcinogenesis. 2006;27:454–64.CrossRefPubMedGoogle Scholar
  60. 60.
    Specht S, Isse K, Nozaki I, Lunz 3rd JG, Demetris AJ. Sprr2a expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis. Clin Exp Metastasis. 2013;30:877–90.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Walaiporn Khansaard
    • 1
  • Anchalee Techasen
    • 2
  • Nisana Namwat
    • 1
  • Puangrat Yongvanit
    • 1
  • Narong Khuntikeo
    • 3
  • Anucha Puapairoj
    • 4
  • Watcharin Loilome
    • 1
  1. 1.Department of Biochemistry, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  2. 2.Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical ScienceKhon Kaen UniversityKhon KaenThailand
  3. 3.Department of Surgery, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  4. 4.Department of Pathology and Liver Fluke and Cholangiocarcinoma Research Center, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand

Personalised recommendations