Cellular Oncology

, Volume 41, Issue 2, pp 169–184 | Cite as

Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma

  • Mauro Montalbano
  • Cristiana Rastellini
  • Joshua T. McGuire
  • Janika Prajapati
  • Ali Shirafkan
  • Renza Vento
  • Luca Cicalese
Original Paper



Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined.


Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed.


We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70 kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40 kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes.


Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.


Hepatocellular carcinoma (HCC) Cirrhotic liver Glypican-3 (GPC3) Furin-Convertase siRNA silencing 



α-smooth muscle actin


bovine serum albumin


cirrhotic distal tissue


hepatocytes from distal cirrhotic liver


Cyclin Kinase Inhibitor


cirrhotic proximal tissue


hepatocytes from proximal liver tissue


extracellular matrix


fetal bovine serum




hepatitis B virus


hepatocellular carcinoma


hepatitis C virus


hepatic stellate cell


heparan sulphate proteoglycan




liver transplantation


non-alcoholic fatty liver disease/non-alcoholic steatohepatitis


primary human hepatocytes




Compliance with ethical standards

Conflict of interest


Supplementary material

13402_2017_364_MOESM1_ESM.tif (1.4 mb)
Supplemental Figure 1 (A) Schematic representation of protocol used to isolate hepatocytes from human liver specimen. (B) Morphologic features of cultured cells obtained from HCC, CP, CD and NL tissues. Cells in primary culture at time 0, 1st and 8th passages. All primary cells proliferate in-vitro maintaining a shape hepatocytes-like in NL- and CD-Hep while showed a spindle-like shape in CP-Hep and HCC. At 8th passages were observed aggregates in CP-Hep and HCC cultures (white bar: 100 μm, magnification: 100X). (TIFF 1435 kb)
13402_2017_364_Fig7_ESM.gif (117 kb)

High resolution image (GIF 117 kb)

13402_2017_364_MOESM2_ESM.tif (6.7 mb)
Supplemental Figure 2 (A) GPC-3 immunofluorescence in margin human CP-HCC tissue. (B) Co-immunofluorescence of N-GPC3 and C-GPC3 with β-Tubulin confirmed that co-localized, while C-GPC3 expressed in the central area of the cell has not interaction with microtubule system in CP-Hep. In HCC cells N- and C-terminal colocalize in the cell and showed an overlap with β-Tubulin (white bar: 200 μm, magnification: 100X). (TIFF 6829 kb)
13402_2017_364_Fig8_ESM.gif (138 kb)

High resolution image (GIF 137 kb)


  1. 1.
    SEER Cancer Statistics Factsheets: Liver and intrahepatic bile duct cancer. National Cancer Institute Bethesda,
  2. 2.
    W.T. London, K.A. McGlynn, in Cancer epidemiology and prevention, 3rd edn., ed. by D. Schottenfeld, J. F. Fraumeni Jr.. Liver cancer (Oxford University Press, New York, 2006), pp. 763–786CrossRefGoogle Scholar
  3. 3.
    S. Mittal, H.B. El-Serag, Epidemiology of hepatocellular carcinoma: Consider the population. J Clin Gastroenterol 47(Suppl), S2–S6 (2013). CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    F.H. Shek, R. Luo, B.Y.H. Lam, W.K. Sung, T.W. Lam, J.M. Luk, M.S. Leung, K.T. Chan, H.K. Wang, C.M. Chan, R.T. Poon, N.P. Lee, Serine peptidase inhibitor Kazal type 1 (SPINK1) as novel downstream effector of the cadherin-17/β-catenin axis in hepatocellular carcinoma. Cell Oncol 40, 443–456 (2017).
  5. 5.
    V. Ramesh, K. Selvarasu, J. Pandian, S. Myilsamy, C. Shanmugasundaram, K. Ganesan, NFκB activation demarcates a subset of hepatocellular carcinoma patients for targeted therapy. Cell Oncol 39, 523–536 (2016).
  6. 6.
    J. Liu, X. Wei, Y. Wu, Y. Wang, Y. Qiu, J. Shi, H. Zou, M. Shao, L. Yu, L. Tong, Giganteaside D induces ROS-mediated apoptosis in human hepatocellular carcinoma cells through the MAPK pathway. Cell Oncol 39, 333–342 (2016).
  7. 7.
    N. Yamauchi, A. Watanabe, M. Hishinuma, K. Ohashi, Y. Midorikawa, Y. Morishita, T. Niki, J. Shibahara, M. Mori, M. Makuuchi, Y. Hippo, T. Kodoma, H. Iwanari, H. Aburatani, M. Fukayama, The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol 18, 1591–1598 (2005).
  8. 8.
    T. Nakatsura, Y. Yoshitake, S. Senju, M. Monji, H. Komori, Y. Motomura, S. Hosaka, T. Beppu, T. Ishiko, H. Kamohara, H. Ashihara, T. Katagiri, Y. Furukawa, S. Fujiyama, M. Oqawa, Y. Nakamura, Y. Nashimura, Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker. Biochem Biophys Res Commun 306, 16–25 (2003).
  9. 9.
    I.P. Chen, S. Ariizumi, M. Nakano, M. Yamamoto, Positive glypican-3 expression in early hepatocellular carcinoma predicts recurrence after hepatectomy. J Gastroenterol 49, 117–125 (2014).
  10. 10.
    B. Yan, J.J. Wei, Y.M. Qian, X.L. Zhao, W.W. Zhang, X. AM, S.H. Zhang, Expression and clinicopathologic significance of glypican 3 in hepatocellular carcinoma. Ann Diagn Pathol 15, 162–169 (2011).
  11. 11.
    Y. Hippo, K. Watanabe, A. Watanabe, Y. Midorikawa, S. Yamamoto, S. Ihara, S. Tokita, H. Iwanari, Y. Ito, K. Nakano, J. Nezu, H. Tsunoda, T. Yoshino, I. Ohizumi, M. Tsuchiya, S. Ohnishi, M. Makuuchi, T. Hamakubo, T. Kodama, H. Aburatani, Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res 64, 2418–2423 (2004).
  12. 12.
    J. Filmus, M. Capurro, J. Rast, Glypicans. Genome Biol 9, 224 (2008).
  13. 13.
    M. Montalbano, C. Rastellini, X. Wang, T. Corsello, M.A. Eltorky, R. Vento, L. Cicalese, Transformation of primary human hepatocytes in hepatocellular carcinoma. Int J Oncol 48, 1205–1217 (2016).
  14. 14.
    M. Montalbano, G. Curcurù, A. Shirafkan, R. Vento, C. Rastellini, L. Cicalese, Modeling of hepatocytes proliferation isolated from proximal and distal zones from human hepatocellular carcinoma lesion. PLoS One 11, e0153613 (2016).
  15. 15.
    J. Filmus, Glypicans in growth control and cancer. Glycobiology 11, 19R–23R (2001).
  16. 16.
    M. Veugelers, B. De Cat, H. Ceulemans, A.M. Bruystens, C. Coomans, J. Durr, J. Vermeesch, P. Marynen, G. David, Glypican-6, a new member of the glypican family of cell surface proteoglycans. J Biol Chem 274, 26968–26977 (1999).
  17. 17.
    M. Ho, Advances in liver cancer antibody therapies: A focus on glypican-3 and mesothelin. BioDrugs 25, 215–222 (2011)CrossRefGoogle Scholar
  18. 18.
    Q. Lin, L.W. Xiong, X.F. Pan, J.F. Gen, G.L. Bao, H.F. Sha, J.X. Feng, C.Y. Ji, M. Chen, Expression of GPC3 protein and its significance in lung squamous cell carcinoma. Med Oncol 29, 663–669 (2012).
  19. 19.
    T. Mounajjed, L. Zhang, W. TT, Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms. Hum Pathol 44, 542–550 (2013).
  20. 20.
    H. Komori, T. Beppu, H. Baba, T. Nakatsura, Y. Nishimura, Assessment of serum GPC3 as a tumor marker for hepatocellular carcinoma and malignant melanoma. Nihon Rinsho 68(Suppl 7), 833–836 (2010)PubMedGoogle Scholar
  21. 21.
    M.I. Capurro, Y.Y. Xiang, C. Lobe, J. Filmus, Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling. Cancer Res 16, 41201–41206 (2005)Google Scholar
  22. 22.
    L. Li, R. Jin, X. Zhang, F. Lv, L. Liu, D. Liu, K. Liu, N. Li, D. Chen, Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma. Hepatology 56, 1380–1390 (2012).
  23. 23.
    W. Gao, M. Ho, The role of glypican-3 in regulating Wnt in hepatocellular carcinomas. Cancer Rep 1, 14–19 (2011)Google Scholar
  24. 24.
    W. Cheng, C.J. Tseng, T.T. Lin, I. Cheng, H.W. Pan, H.C. Hsu, Y.M. Lee, Glypican-3-mediated oncogenesis involves the Insulinlike growth factor-signaling pathway. Carcinogenesis 29, 1319–1326 (2008).
  25. 25.
    S. Liu, Y. Li, W. Chen, P. Zheng, T. Liu, W. He, J. Zhang, X. Zeng, Silencing glypican-3 expression induces apoptosis in human hepatocellular carcinoma cells. Biochem Biophys Res Commun 419, 656–661 (2012).
  26. 26.
    B. Le Bail, G. Belleannee, P.H. Bernard, J. Saric, Balabaud C, Bioulac-sage P’ adenomatous hyperplasia in cirrhotic livers: Histological evaluation, cellular density, and proliferative activity of 35 macro nodular lesions in the cirrhotic explants of 10 adult French patients. Hum Pathol 26, 897–906 (1995).
  27. 27.
    M. Sakamoto, S. Hirohashi, Shimosato Y’ early stages of multistep hepatocarcinogenesis: Adenomatous hyperplasia and early hepatocellular carcinoma. Hum Pathol 22, 172–178 (1991).
  28. 28.
    T. Takayama, T. Kosuge, S. Yamazaki, H. Hasegawa, N. Okazaki, K. Takayasu, S. Hirohashi, M. Sakamoto, M. Makuuchi, Y. Motoo, Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma. Lancet 336, 1150–1153 (1990).
  29. 29.
    M. Borzio, S. Bruno, M. Roncalli, G.C. Mels, G. Ramella, F. Borzio, G. Leandro, E. Servida, M. Podda, Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: A prospective study. Gastroenterology 108, 812–817 (1995).
  30. 30.
    C. Cohen, S.D. Berson, E.W. Geddes, Liver cell dysplasia: Association with hepatocellular carcinoma, cirrhosis and hepatitis B antigen carrier status. Cancer 44, 1671–1676 (1979).<1671::AID-CNCR2820440521>3.0.CO;2-Y
  31. 31.
    B. Le Bail, C. Balabaud, Prevalence of liver cell dysplasia and association with HCC in a series of 100 cirrhotic liver explants. J Hepatol 27, 835–842 (1997).
  32. 32.
    X.Y. Wang, F. Degos, S. Dubois, S. Tessiore, M. Allegretta, R.D. Guttmann, S. Jothy, J. Belghiti, P. Bedossa, V. Paradis, Glypican-3 expression in hepatocellular tumors: Diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Hum Pathol 37, 1435–1441 (2006).
  33. 33.
    L. Libbrecht, T. Severi, D. Casiman, S. Vander Borght, J. Pirenne, Nevens F, Verslype C, van pelt J, Roskams T, Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol 30, 1405–1411 (2006).
  34. 34.
    Y.H. Huang, K.H. Lin, C.H. Liao, M.W. Lai, Y.H. Tseng, C.T. Yeh, Furin overexpression suppresses tumor growth and predicts a better postoperative disease-free survival in hepatocellular carcinoma. PLoS One 7, e40738 (2012).
  35. 35.
    W. Sang, W. Zhang, W. Cui, X. Li, G. Abulajiang, Q. Li, Arginase-1 is a more sensitive marker than HepPar-1 and AFP in differential diagnosis of hepatocellular carcinoma from non-hepatocellular carcinoma. Tumor Biol 36, 3881–3886 (2015).
  36. 36.
    J. Hu, L. Che, L. Li, M.G. Pilo, A. Cigliano, S. Ribback, X. Li, G. Latte, M. Mela, M. Evert, F. Dombrowski, G. Zheng, X. Chen, D.F. Calvisi, Co-activation of AKT and c-Met triggers rapid hepatocellular carcinoma development via the mTORC1/FASN pathway in mice. Sci Rep 6, 20484 (2016).
  37. 37.
    Y. Matsuda, S. Yamagiwa, M. Takamura, Y. Honda, Y. Ishimoto, T. Ichida, Y. Aoyagi, Overexpressed Id-1 is associated with a high risk of hepatocellular carcinoma development in patients with cirrhosis without transcriptional repression of p16. Cancer 104, 1037–1044 (2005).
  38. 38.
    Jin X, Liu Y, Liu J, Lu W, Liang Z, Zhang D, Liu G, Zhu H, Xu N, Liang S, The overexpression of IQGAP1 and β-Catenin is associated with tumor progression in hepatocellular carcinoma in vitro and in vivo. PLoS One 10, e0133770 (2015)Google Scholar
  39. 39.
    A. Kakehashi, M. Inoue, M. Wei, S. Fukushima, H. Wanibuchi, Cytokeratin 8/18 overexpression and complex formation as an indicator of GST-P positive foci transformation into hepatocellular carcinomas. Toxicol Appl Pharmacol 238, 71–79 (2009).
  40. 40.
    Y.X. Bao, Q. Cao, Y. Yang, R. Mao, L. Xiao, H. Zhang, H.R. Zhao, H. Wen, Expression and prognostic significance of golgiglycoprotein73 (GP73) with epithelial-mesenchymal transition (EMT) related molecules in hepatocellular carcinoma (HCC). Diagn Pathol 8, 197 (2013).
  41. 41.
    M. Capurro, W. Shi, T. Izumikawa, H. Kitagawa, J. Filmus, Processing by convertases is required for glypican-3-induced inhibition of Hedgehog signaling. J Biol Chem 290, 7576–7585 (2015).

Copyright information

© International Society for Cellular Oncology 2017

Authors and Affiliations

  • Mauro Montalbano
    • 1
    • 2
  • Cristiana Rastellini
    • 1
  • Joshua T. McGuire
    • 1
  • Janika Prajapati
    • 1
  • Ali Shirafkan
    • 1
  • Renza Vento
    • 3
    • 4
  • Luca Cicalese
    • 1
  1. 1.Department of SurgeryUniversity of Texas Medical BranchGalvestonUSA
  2. 2.Center of Biomedical EngineeringUniversity of Texas Medical BranchGalvestonUSA
  3. 3.Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, PolyclinicUniversity of PalermoPalermoItaly
  4. 4.Institute for Cancer Research and Molecular Medicine and Center of Biotechnology, College of Science and BiotechnologyTemple UniversityPhiladelphiaUSA

Personalised recommendations