Skip to main content

Advertisement

SpringerLink
Log in

Epigenetic silencing of sFRP1 activates the canonical Wnt pathway and contributes to increased cell growth and proliferation in hepatocellular carcinoma

  • Research Article
  • Published:
Tumor Biology

Abstract

The Wnt pathway is a key regulator of embryonic development and stem cells, and its aberrant activation is associated with human malignancies, most notably hepatocellular carcinoma (HCC). Epigenetic deregulation of the genes encoding the secreted frizzled-related proteins (sFRPs), the Wnt signalling antagonists, has been linked with aberrant hyperactivation of the Wnt signalling in HCC cells; however, the precise underlying mechanism remains elusive. We investigated the methylation profiles of Wnt antagonists in liver samples of different stages of HCC development and liver cancer cell lines and studied the functional impact of aberrant epigenetic silencing of sFRPs on the canonical Wnt pathway and cell viability. We found that the sFRP1 gene encoding the subunit is a frequent target of aberrant DNA hypermethylation and silencing in HCC tumours, whereas other extracellular Wnt antagonists, WIF1 and Dkk3, exhibited no methylation in tumour cells, consistent with the notion that aberrant methylation events in cancer cells are non-randomly distributed among the genes and that there is a strong preference for hypermethylation of specific genes in HCC. In addition, by comparing sFRP1 methylation status in HCC tumours with normal, cirrhotic and chronic hepatitis liver tissues, we identified sFRP1 gene as a potential early marker of HCC. The restoration of sFRP1 expression in cancer cells by ectopic expression inhibited Wnt activity accompanied with destabilization of β-catenin and downregulation of c-Myc and cyclin D1, the known downstream targets of Wnt pathway. Importantly, restoring sFRP1 levels in cancer cells inhibited cell growth and induced apoptotic cell death. This study supports the critical role for sFRP1 silencing in hepatocellular carcinoma and reinforces the importance of the Wnt antagonists in preventing oncogenic stabilization of β-catenin and chronic activation of the canonical Wnt pathway, suggesting that sFRP1 may be an attractive target for early cancer detection and therapeutic intervention.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

HCC:

Hepatocellular carcinoma

HBV:

Hepatitis B virus

HCV:

Hepatitis C virus

DMEM:

Dulbecco’s minimum essential medium

RT-QPCR:

Real-time quantitative transcription PCR

sFRPs:

Secreted frizzled-related proteins

WIF1:

Wnt inhibitory factor 1

Dkk3:

Dickkopf 3

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

References

  1. Globocan (2008) International Agency for Research on Cancer, Lyon, France

  2. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 2006;6(2):107–16.

    Article  PubMed  CAS  Google Scholar 

  3. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92.

    Article  PubMed  CAS  Google Scholar 

  4. Herceg Z, Paliwal A. Epigenetic mechanisms in hepatocellular carcinoma: how environmental factors influence the epigenome. Mutat Res. 2011;727(3):55–61.

    Article  PubMed  CAS  Google Scholar 

  5. Polakis P. Wnt signaling and cancer. Genes Dev. 2000;14(15):1837–51.

    PubMed  CAS  Google Scholar 

  6. de La Coste A, Romagnolo B, Billuart P, Renard CA, Buendia MA, Soubrane O, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci U S A. 1998;95(15):8847–51.

    Article  Google Scholar 

  7. Miyoshi Y, Iwao K, Nagasawa Y, Aihara T, Sasaki Y, Imaoka S, et al. Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res. 1998;58(12):2524–7.

    PubMed  CAS  Google Scholar 

  8. Huang H, Fujii H, Sankila A, Mahler-Araujo BM, Matsuda M, Cathomas G, et al. Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am J Pathol. 1999;155(6):1795–801.

    Article  PubMed  CAS  Google Scholar 

  9. Chien AJ, Conrad WH, Moon RT. A Wnt survival guide: from flies to human disease. J Invest Dermatol. 2009;129(7):1614–27.

    Article  PubMed  CAS  Google Scholar 

  10. Jones SE, Jomary C. Secreted frizzled-related proteins: searching for relationships and patterns. Bioessays. 2002;24(9):811–20.

    Article  PubMed  CAS  Google Scholar 

  11. Chong JM, Uren A, Rubin JS, Speicher DW. Disulfide bond assignments of secreted frizzled-related protein-1 provide insights about frizzled homology and netrin modules. J Biol Chem. 2002;277(7):5134–44.

    Article  PubMed  CAS  Google Scholar 

  12. Caldwell GM, Jones C, Gensberg K, Jan S, Hardy RG, Byrd P, et al. The Wnt antagonist sFRP1 in colorectal tumorigenesis. Cancer Res. 2004;64(3):883–8.

    Article  PubMed  CAS  Google Scholar 

  13. Tanaka J, Watanabe T, Kanazawa T, Tada T, Kazama Y, Tanaka T, et al. Silencing of secreted frizzled-related protein genes in MSI colorectal carcinogenesis. Hepatogastroenterology. 2008;55(85):1265–8.

    PubMed  CAS  Google Scholar 

  14. Qi J, Zhu YQ, Luo J, Tao WH. Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor. World J Gastroenterol. 2006;12(44):7113–7.

    PubMed  CAS  Google Scholar 

  15. Zou H, Molina JR, Harrington JJ, Osborn NK, Klatt KK, Romero Y, et al. Aberrant methylation of secreted frizzled-related protein genes in esophageal adenocarcinoma and Barrett’s esophagus. Int J Cancer. 2005;116(4):584–91.

    Article  PubMed  CAS  Google Scholar 

  16. Clement G, Braunschweig R, Pasquier N, Bosman FT, Benhattar J. Alterations of the Wnt signaling pathway during the neoplastic progression of Barrett’s esophagus. Oncogene. 2006;25(21):3084–92.

    Article  PubMed  CAS  Google Scholar 

  17. Stoehr R, Wissmann C, Suzuki H, Knuechel R, Krieg RC, Klopocki E, et al. Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer. Lab Invest. 2004;84(4):465–78.

    Article  PubMed  CAS  Google Scholar 

  18. Urakami S, Shiina H, Enokida H, Kawakami T, Kawamoto K, Hirata H, et al. Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection. Clin Cancer Res. 2006;12(7 Pt 1):2109–16.

    Article  PubMed  CAS  Google Scholar 

  19. Nojima M, Suzuki H, Toyota M, Watanabe Y, Maruyama R, Sasaki S, et al. Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer. Oncogene. 2007;26(32):4699–713.

    Article  PubMed  CAS  Google Scholar 

  20. Zhao CH, Bu XM, Zhang N. Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer. World J Gastroenterol. 2007;13(15):2214–7.

    PubMed  CAS  Google Scholar 

  21. Morris MR, Ricketts C, Gentle D, Abdulrahman M, Clarke N, Brown M, et al. Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma. Oncogene. 2010;29(14):2104–17.

    Article  PubMed  CAS  Google Scholar 

  22. Costa VL, Henrique R, Ribeiro FR, Carvalho JR, Oliveira J, Lobo F, et al. Epigenetic regulation of Wnt signaling pathway in urological cancer. Epigenetics. 2010;5(4):343–51.

    Article  PubMed  CAS  Google Scholar 

  23. Fukui T, Kondo M, Ito G, Maeda O, Sato N, Yoshioka H, et al. Transcriptional silencing of secreted frizzled related protein 1 (SFRP 1) by promoter hypermethylation in non-small-cell lung cancer. Oncogene. 2005;24(41):6323–7.

    Article  PubMed  CAS  Google Scholar 

  24. Tang M, Torres-Lanzas J, Lopez-Rios F, Esteller M, Sanchez-Cespedes M. Wnt signaling promoter hypermethylation distinguishes lung primary adenocarcinomas from colorectal metastasis to the lung. Int J Cancer. 2006;119(11):2603–6.

    Article  PubMed  CAS  Google Scholar 

  25. Shih YL, Hsieh CB, Lai HC, Yan MD, Hsieh TY, Chao YC, et al. SFRP1 suppressed hepatoma cells growth through Wnt canonical signaling pathway. Int J Cancer. 2007;121(5):1028–35.

    Article  PubMed  CAS  Google Scholar 

  26. Takagi H, Sasaki S, Suzuki H, Toyota M, Maruyama R, Nojima M, et al. Frequent epigenetic inactivation of SFRP genes in hepatocellular carcinoma. J Gastroenterol. 2008;43(5):378–89.

    Article  PubMed  Google Scholar 

  27. Murr R, Loizou JI, Yang YG, Cuenin C, Li H, Wang ZQ, et al. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat Cell Biol. 2006;8(1):91–9.

    Article  PubMed  CAS  Google Scholar 

  28. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science. 1997;275(5307):1784–7.

    Article  PubMed  CAS  Google Scholar 

  29. Finkbeiner MG, Sawan C, Ouzounova M, Murr R, Herceg Z. HAT cofactor TRRAP mediates beta-catenin ubiquitination on the chromatin and the regulation of the canonical Wnt pathway. Cell Cycle. 2008;7(24):3908–14.

    Article  PubMed  CAS  Google Scholar 

  30. Hecht A, Vleminckx K, Stemmler MP, van Roy F, Kemler R. The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J. 2000;19(8):1839–50.

    Article  PubMed  CAS  Google Scholar 

  31. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997;16(13):3797–804.

    Article  PubMed  CAS  Google Scholar 

  32. Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med. 2011;17(3):330–9.

    Article  PubMed  CAS  Google Scholar 

  33. Herceg Z. Epigenetics and cancer: towards an evaluation of the impact of environmental and dietary factors. Mutagenesis. 2007;22(2):91–103.

    Article  PubMed  CAS  Google Scholar 

  34. Esteller M. Aberrant DNA methylation as a cancer-inducing mechanism. Annu Rev Pharmacol Toxicol. 2005;45:629–56.

    Article  PubMed  CAS  Google Scholar 

  35. Agrawal A, Murphy RF, Agrawal DK. DNA methylation in breast and colorectal cancers. Mod Pathol. 2007;20(7):711–21.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

P. Kaur and S. Mani are supported by a post-doctoral fellowship from the International Agency for Research on Cancer (IARC), Lyon, France. This work in the IARC Epigenetics Group is supported by grants from l’Agence Nationale de Recherhe Contre le Sida et Hépatites Virales (ANRS, France); the Association pour la Recherche sur le Cancer (ARC, France); la Ligue Nationale (Française) Contre le Cancer (France); and the Swiss Bridge Award.

Financial disclosure

This work was supported by la Ligue Nationale (Française) Contre le Cancer, l’Association pour le Recherche Contre le Cancer (l’ARC), l’Agence Nationale de Recherche Contre le Sida et Hépatites Virales (ANRS, France) and Swiss Bridge Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Epigenetics Group, International Agency for Research on Cancer (IARC), 150 Cours Albert-Thomas, 69008, Lyon, France

    Pushpinder Kaur, Samson Mani, Marie-Pierre Cros & Zdenko Herceg

  2. Molecular Carcinogenesis Group, International Agency for Research on Cancer, 69008, Lyon, France

    Pierre Hainaut

  3. Service d’Anatomie Pathologique, Edouard Herriot Hospital, Lyon, France

    Jean-Yves Scoazec

  4. INSERM, U871, 69003, Lyon, France

    Isabelle Chemin

Authors
  1. Pushpinder Kaur
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samson Mani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marie-Pierre Cros
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean-Yves Scoazec
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Isabelle Chemin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pierre Hainaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zdenko Herceg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zdenko Herceg.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 71 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kaur, P., Mani, S., Cros, MP. et al. Epigenetic silencing of sFRP1 activates the canonical Wnt pathway and contributes to increased cell growth and proliferation in hepatocellular carcinoma. Tumor Biol. 33, 325–336 (2012). https://doi.org/10.1007/s13277-012-0331-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-012-0331-5

Keywords

Search

Navigation