Journal of Gastroenterology

, Volume 49, Issue 1, pp 81–92 | Cite as

C-ERC/mesothelin provokes lymphatic invasion of colorectal adenocarcinoma

  • Futoshi Kawamata
  • Shigenori Homma
  • Hirofumi Kamachi
  • Takahiro Einama
  • Yasutaka Kato
  • Masumi Tsuda
  • Shinya Tanaka
  • Masahiro Maeda
  • Kazunori Kajino
  • Okio Hino
  • Norihiko Takahashi
  • Toshiya Kamiyama
  • Hiroshi NishiharaEmail author
  • Akinobu Taketomi
  • Satoru Todo
Original Article—Alimentary Tract



Lymph node metastasis is a key event of colorectal cancer (CRC) progression. Mesothelin is expressed in various types of malignant tumor and associated with an unfavorable prognosis. The full-length mesothelin (Full-ERC) is cleaved by protease into membrane-bound C-ERC/mesothelin and N-ERC/mesothelin which is secreted into the blood. The aim of this study was to examine the biological role of mesothelin in CRC by clinicopathological analysis and in vitro lymphatic invasion assay.


Ninety-one cases of CRC specimens were immunohistochemically examined and the localization of mesothelin in luminal membrane and/or cytoplasm was also evaluated. Lymphatic invasion assay was also performed using the human CRC cell line, WiDr, which was transfected with Full-, N- and C-ERC/mesothelin expression plasmids (Full-WiDr, N-WiDr and C-WiDr).


Immunohistochemically, “luminal membrane positive” of mesothelin was identified in 37.4 %, and correlated with lymphatic permeation and lymph node metastasis, but not with patients’ prognosis. Interestingly, among the patients with lymph node metastasis (N = 38), “luminal membrane positive” of mesothelin significantly correlated with unfavorable patients’ outcome. In addition, lymphatic invasion assay revealed that Full-WiDr and C-WiDr more significantly invaded human lymphatic endothelial cells than the Mock-WiDr (P < 0.01).


The luminal membrane expression of mesothelin was associated with unfavorable prognosis of CRC patients with lymph node metastasis. Moreover, this is the first report to prove the biological function of C-ERC/mesothelin associated with lymphatic invasion of cancer in vitro.


C-ERC/mesothelin Colorectal cancer Lymphatic invasion 



This research was supported by a Grant-in-Aid for Scientific Research (KAKENHI) and by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

535_2013_773_MOESM1_ESM.tif (102 kb)
Supplemental Figure 1. Scheme of lymphatic invasion and adhesion assay. (a): Modified transwell invasion assays. 5 x 104 of GFP-expressing Full-WiDr, N-WiDr, C-WiDr or Mock-WiDr were loaded on the top of the upper side of transwell inserts which were covered with a confluent monolayer of hLEC, and invaded cells were counted on the bottom side of the inserts. (b): Modified adhesion assays. 1 x 105 of hLEC were plated on a 12-well dish and incubated for 3 days. After confirmation of hLEC in confluence and monolayer by microscope, 1 x 105 of GFP-expressing Full-WiDr, N-WiDr, C-WiDr or Mock-WiDr were resuspended and plated on the cell layer of hLEC. After the incubation at 37°C for 30 min, non-adherent or loosely attached cells were removed by gently washing the wells twice with PBS. The attached GFP-expressing cells (Full-WiDr, N-WiDr, C-WiDr and Mock-WiDr) on hLEC were counted using a fluorescence microscope. hLEC: human lymphatic endothelial cells (TIFF 102 kb)
535_2013_773_MOESM2_ESM.tif (76 kb)
Supplemental Figure 2. Expression of mesothelin and patients’ prognosis (a, c): “High-level expression” and “cytoplasmic expression” of mesothelin were not correlated with total patients’ prognosis (N = 91, P = 0.26, P = 0.31). (b, d): Among the patients with lymph node metastasis (N = 38), “high-level expression” and “cytoplasmic expression” of mesothelin were not correlated with patients’ prognosis (P = 0.73, P = 0.89) (TIFF 75 kb)
535_2013_773_MOESM3_ESM.docx (24 kb)
Supplemental Table 1 (DOCX 24 kb)
535_2013_773_MOESM4_ESM.docx (16 kb)
Supplemental Table 2 (DOCX 16 kb)


  1. 1.
    Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10–29.PubMedCrossRefGoogle Scholar
  2. 2.
    Kotake K, Honjo S, Sugihara K, Kato T, Kodaira S, Takahashi T, et al. Changes in colorectal cancer during a 20-year period: an extended report from the multi-institutional registry of large bowel cancer, Japan. Dis Colon Rectum. 2003;46:S32–43.PubMedGoogle Scholar
  3. 3.
    Watanabe T, Itabashi M, Shimada Y, Tanaka S, Ito Y, Ajioka Y, et al. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2010 for the treatment of colorectal cancer. Int J Clin Oncol. 2012;17:1–29.PubMedCrossRefGoogle Scholar
  4. 4.
    Nitsche U, Maak M, Schuster T, Kunzli B, Langer R, Slotta-Huspenina J, et al. Prediction of prognosis is not improved by the seventh and latest edition of the TNM classification for colorectal cancer in a single-center collective. Ann Surg. 2011;254:793–800.PubMedCrossRefGoogle Scholar
  5. 5.
    Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2000;18:136–47.PubMedGoogle Scholar
  6. 6.
    Verdecchia A, Francisci S, Brenner H, Gatta G, Micheli A, Mangone L, et al. Recent cancer survival in Europe: a 2000–02 period analysis of EUROCARE-4 data. Lancet Oncol. 2007;8:784–96.PubMedCrossRefGoogle Scholar
  7. 7.
    Newland RC, Chapuis PH, Pheils MT, MacPherson JG. The relationship of survival to staging and grading of colorectal carcinoma: a prospective study of 503 cases. Cancer. 1981;47:1424–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Akagi T, Hijiya N, Inomata M, Shiraishi N, Moriyama M, Kitano S. Visinin-like protein-1 overexpression is an indicator of lymph node metastasis and poor prognosis in colorectal cancer patients. Int J Cancer. 2012;131:1307–17.PubMedCrossRefGoogle Scholar
  9. 9.
    Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci USA. 1996;93:136–40.PubMedCrossRefGoogle Scholar
  10. 10.
    Chang K, Pastan I, Willingham MC. Isolation and characterization of a monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium. Int J Cancer. 1992;50:373–81.PubMedCrossRefGoogle Scholar
  11. 11.
    Argani P, Iacobuzio-Donahue C, Ryu B, Rosty C, Goggins M, Wilentz RE, et al. Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE). Clin Cancer Res. 2001;7:3862–8.PubMedGoogle Scholar
  12. 12.
    Hassan R, Kreitman RJ, Pastan I, Willingham MC. Localization of mesothelin in epithelial ovarian cancer. Appl Immunohistochem Mol Morphol. 2005;13:243–7.PubMedCrossRefGoogle Scholar
  13. 13.
    Ordonez NG. Value of mesothelin immunostaining in the diagnosis of mesothelioma. Mod Pathol. 2003;16:192–7.PubMedCrossRefGoogle Scholar
  14. 14.
    Ordonez NG. Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol. 2003;27:1418–28.PubMedCrossRefGoogle Scholar
  15. 15.
    Einama T, Kamachi H, Nishihara H, Homma S, Kanno H, Takahashi K, et al. Co-expression of mesothelin and CA125 correlates with unfavorable patient outcome in pancreatic ductal adenocarcinoma. Pancreas. 2011;40:1276–82.PubMedCrossRefGoogle Scholar
  16. 16.
    Hassan R, Bera T, Pastan I. Mesothelin: a new target for immunotherapy. Clin Cancer Res. 2004;10:3937–42.PubMedCrossRefGoogle Scholar
  17. 17.
    Wang K, Bodempudi V, Liu Z, Borrego-Diaz E, Yamoutpoor F, Meyer A, et al. Inhibition of mesothelin as a novel strategy for targeting cancer cells. PloS one. 2012;7:e33214.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation. Carcinogenesis. 2011;32:1013–24.PubMedCrossRefGoogle Scholar
  19. 19.
    Bharadwaj U, Marin-Muller C, Li M, Chen C, Yao Q. Mesothelin confers pancreatic cancer cell resistance to TNF-alpha-induced apoptosis through Akt/PI3K/NF-kappaB activation and IL-6/Mcl-1 overexpression. Mol Cancer. 2011;10:106.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Li M, Bharadwaj U, Zhang R, Zhang S, Mu H, Fisher WE, et al. Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer. Mol Cancer Ther. 2008;7:286–96.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Servais EL, Colovos C, Rodriguez L, Bograd AJ, Nitadori J, Sima C, et al. Mesothelin overexpression promotes mesothelioma cell invasion and mmp-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin Cancer Res. 2012;18:2478–89.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Einama T, Homma S, Kamachi H, Kawamata F, Takahashi K, Takahashi N, et al. Luminal membrane expression of mesothelin is a prominent poor prognostic factor for gastric cancer. Br J Cancer. 2012;107:137–42.PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Liebig B, Brabletz T, Staege MS, Wulfanger J, Riemann D, Burdach S, et al. Forced expression of deltaN-TCF-1B in colon cancer derived cell lines is accompanied by the induction of CEACAM5/6 and mesothelin. Cancer Lett. 2005;223:159–67.PubMedCrossRefGoogle Scholar
  24. 24.
    Sobin LH, Wittekind C. TNM classification of malignant tumors. 6th ed. New York: Wiley-Liss; 2002.Google Scholar
  25. 25.
    Wang T, Kajino K, Abe M, Tan K, Maruo M, Sun G, et al. Suppression of cell death by the secretory form of N-terminal ERC/mesothelin. Int J Mol Med. 2010;26:185–91.PubMedGoogle Scholar
  26. 26.
    Akagi T, Sasai K, Hanafusa H. Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation. Proc Natl Acad Sci USA. 2003;100:13567–72.PubMedCrossRefGoogle Scholar
  27. 27.
    Ren YR, Patel K, Paun BC, Kern SE. Structural analysis of the cancer-specific promoter in mesothelin and in other genes overexpressed in cancers. J Biol Chem. 2011;286:11960–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Shiomi K, Miyamoto H, Segawa T, Hagiwara Y, Ota A, Maeda M, et al. Novel ELISA system for detection of N-ERC/mesothelin in the sera of mesothelioma patients. Cancer Sci. 2006;97:928–32.PubMedCrossRefGoogle Scholar
  29. 29.
    Ishikawa K, Segawa T, Hagiwara Y, Maeda M, Abe M, Hino O. Establishment of novel mAb to human ERC/mesothelin useful for study and diagnosis of ERC/mesothelin-expressing cancers. Pathol Int. 2009;59:161–6.PubMedCrossRefGoogle Scholar
  30. 30.
    Inami K, Abe M, Takeda K, Hagiwara Y, Maeda M, Segawa T, et al. Antitumor activity of anti-C-ERC/mesothelin monoclonal antibody in vivo. Cancer Sci. 2010;101:969–74.PubMedCrossRefGoogle Scholar
  31. 31.
    Cheng WF, Huang CY, Chang MC, Hu YH, Chiang YC, Chen YL, et al. High mesothelin correlates with chemoresistance and poor survival in epithelial ovarian carcinoma. Br J Cancer. 2009;100:1144–53.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Winter JM, Tang LH, Klimstra DS, Brennan MF, Brody JR, Rocha FG, et al. A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers. PloS One. 2012;7:e40157.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Shimizu A, Hirono S, Tani M, Kawai M, Okada K, Miyazawa M, et al. Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma. Cancer Sci. 2012;103:739–46.PubMedCrossRefGoogle Scholar
  34. 34.
    Joshi MB, Ivanov D, Philippova M, Erne P, Resink TJ. Integrin-linked kinase is an essential mediator for T-cadherin-dependent signaling via Akt and GSK3beta in endothelial cells. FASEB J Off Publ Fed Am Soc Exp Biol. 2007;21:3083–95.Google Scholar
  35. 35.
    Philippova M, Ivanov D, Joshi MB, Kyriakakis E, Rupp K, Afonyushkin T, et al. Identification of proteins associating with glycosylphosphatidylinositol- anchored T-cadherin on the surface of vascular endothelial cells: role for Grp78/BiP in T-cadherin-dependent cell survival. Mol Cell Biol. 2008;28:4004–17.PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Garmy-Susini B, Avraamides CJ, Schmid MC, Foubert P, Ellies LG, Barnes L, et al. Integrin alpha4beta1 signaling is required for lymphangiogenesis and tumor metastasis. Cancer Res. 2010;70:3042–51.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Dulak AM, Schumacher SE, van Lieshout J, Imamura Y, Fox C, Shim B, et al. Gastrointestinal adenocarcinomas of the esophagus, stomach, and colon exhibit distinct patterns of genome instability and oncogenesis. Cancer Res. 2012;72:4383–93.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Souglakos J, Ziras N, Kakolyris S, Boukovinas I, Kentepozidis N, Makrantonakis P, et al. Randomised phase-II trial of CAPIRI (capecitabine, irinotecan) plus bevacizumab vs FOLFIRI (folinic acid, 5-fluorouracil, irinotecan) plus bevacizumab as first-line treatment of patients with unresectable/metastatic colorectal cancer (mCRC). Br J Cancer. 2012;106:453–9.PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Alberts SR, Sargent DJ, Nair S, Mahoney MR, Mooney M, Thibodeau SN, et al. Effect of oxaliplatin, fluorouracil, and leucovorin with or without cetuximab on survival among patients with resected stage III colon cancer: a randomized trial. JAMA. 2012;307:1383–93.PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Kreitman RJ, Hassan R, FitzGerald DJ, Pastan I. Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P. Clin Cancer Res. 2009;15:5274–9.PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Hassan R, Cohen SJ, Phillips M, Pastan I, Sharon E, Kelly RJ, et al. Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAb-009 in patients with mesothelin expressing cancers. Clin Cancer Res. 2010;16:6132–8.PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Hassan R, Bullock S, Premkumar A, Kreitman RJ, Kindler H, Willingham MC, et al. Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res. 2007;13:5144–9.PubMedCrossRefGoogle Scholar
  43. 43.
    Hassan R, Ebel W, Routhier EL, Patel R, Kline JB, Zhang J, et al. Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin. Cancer Immun. 2007;7:20.PubMedGoogle Scholar
  44. 44.
    Hassan R, Schweizer C, Lu KF, Schuler B, Remaley AT, Weil SC, et al. Inhibition of mesothelin-CA-125 interaction in patients with mesothelioma by the anti-mesothelin monoclonal antibody MORAb-009: implications for cancer therapy. Lung Cancer. 2010;68:455–9.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Futoshi Kawamata
    • 1
  • Shigenori Homma
    • 1
  • Hirofumi Kamachi
    • 1
  • Takahiro Einama
    • 1
  • Yasutaka Kato
    • 2
  • Masumi Tsuda
    • 2
  • Shinya Tanaka
    • 2
  • Masahiro Maeda
    • 3
  • Kazunori Kajino
    • 4
  • Okio Hino
    • 4
  • Norihiko Takahashi
    • 1
  • Toshiya Kamiyama
    • 1
  • Hiroshi Nishihara
    • 5
    Email author
  • Akinobu Taketomi
    • 1
  • Satoru Todo
    • 1
  1. 1.Department of General SurgeryHokkaido University, Graduate School of MedicineSapporoJapan
  2. 2.Department of Cancer PathologyHokkaido University School of MedicineSapporoJapan
  3. 3.Department of Research and DevelopmentImmuno-Biological Laboratories Co. Ltd.GunmaJapan
  4. 4.Department of Pathology and OncologyJuntendo University School of MedicineTokyoJapan
  5. 5.Department of Translational PathologyHokkaido University, Graduate School of MedicineSapporoJapan

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