Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect

Abstract

Tumor-suppressor genes on chromosome X can be inactivated by a single hit, any of the point mutations, chromosomal loss and aberrant DNA methylation. As aberrant DNA methylation can be induced frequently, we here aimed to identify a tumor-suppressor gene on chromosome X inactivated by promoter DNA methylation. Of 69 genes on chromosome X upregulated by treatment of a gastric cancer cell line with a DNA-demethylating agent, 5-aza-2′-deoxycytidine, 11 genes had low or no expression in the cell line and abundant expression in normal gastric mucosae. Among them, FHL1 was frequently methylation-silenced in gastric and colon cancer cell lines, and methylated in primary gastric (21/80) and colon (5/50) cancers. Knockdown of the endogenous FHL1 in two cell lines by two kinds of shRNAs significantly increased cell growth in vitro and sizes of xenografts in nude mice. Expression of exogenous FHL1 in a non-expressing cell line significantly reduced its migration, invasion and growth. Notably, a somatic mutation (G642T; Lys214Asn) was identified in one of 144 colon cancer specimens, and the mutant FHL1 was shown to lack its inhibitory effects on migration, invasion and growth. FHL1 methylation was associated with Helicobacter pylori infection and accumulated in normal-appearing gastric mucosae of gastric cancer patients. These data showed that FHL1 is a methylation-silenced tumor-suppressor gene on chromosome X in gastrointestinal cancers, and that its silencing contributes to the formation of an epigenetic field for cancerization.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Knudson AG . Two genetic hits (more or less) to cancer. Nat Rev Cancer 2001; 1: 157–162.

    Article  CAS  PubMed Central  Google Scholar 

  2. Ushijima T . Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer 2005; 5: 223–231.

    Article  CAS  Google Scholar 

  3. Jones PA, Baylin SB . The epigenomics of cancer. Cell 2007; 128: 683–692.

    Article  CAS  PubMed Central  Google Scholar 

  4. Rivera MN, Kim WJ, Wells J, Driscoll DR, Brannigan BW, Han M et al. An X chromosome gene, WTX, is commonly inactivated in Wilms tumor. Science 2007; 315: 642–645.

    Article  CAS  Google Scholar 

  5. Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W et al. FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell 2007; 129: 1275–1286.

    Article  CAS  PubMed Central  Google Scholar 

  6. Wang L, Liu R, Li W, Chen C, Katoh H, Chen GY et al. Somatic single hits inactivate the X-linked tumor suppressor FOXP3 in the prostate. Cancer Cell 2009; 16: 336–346.

    Article  CAS  PubMed Central  Google Scholar 

  7. Van Vlierberghe P, Palomero T, Khiabanian H, Van Der Meulen J, Castillo M, Van Roy N et al. PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet 2010; 42: 338–342.

    Article  CAS  PubMed Central  Google Scholar 

  8. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res 2006; 12: 989–995.

    Article  CAS  Google Scholar 

  9. Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M et al. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer 2009; 124: 2367–2374.

    Article  CAS  PubMed Central  Google Scholar 

  10. Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J et al. MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 2005; 97: 1330–1338.

    Article  CAS  Google Scholar 

  11. Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S . Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis—a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma. Hepatology 2000; 32: 970–979.

    Article  CAS  Google Scholar 

  12. Ishii T, Murakami J, Notohara K, Cullings HM, Sasamoto H, Kambara T et al. Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa. Gut 2007; 56: 13–19.

    Article  CAS  Google Scholar 

  13. Oka D, Yamashita S, Tomioka T, Nakanishi Y, Kato H, Kaminishi M et al. The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history: a target for risk diagnosis and prevention of esophageal cancers. Cancer 2009; 115: 3412–3426.

    Article  CAS  Google Scholar 

  14. Lee YC, Wang HP, Wang CP, Ko JY, Lee JM, Chiu HM et al. Revisit of field cancerization in squamous cell carcinoma of upper aerodigestive tract: better risk assessment with epigenetic markers. Cancer Prev Res 2011; 4: 1982–1992.

    Article  CAS  Google Scholar 

  15. Yan PS, Venkataramu C, Ibrahim A, Liu JC, Shen RZ, Diaz NM et al. Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue. Clin Cancer Res 2006; 12: 6626–6636.

    Article  CAS  Google Scholar 

  16. Arai E, Kanai Y, Ushijima S, Fujimoto H, Mukai K, Hirohashi S . Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissues. Int J Cancer 2006; 119: 288–296.

    Article  CAS  Google Scholar 

  17. Nakajima T, Maekita T, Oda I, Gotoda T, Yamamoto S, Umemura S et al. Higher methylation levels in gastric mucosae significantly correlate with higher risk of gastric cancers. Cancer Epidemiol Biomarkers Prev 2006; 15: 2317–2321.

    Article  CAS  Google Scholar 

  18. Ushijima T . Epigenetic field for cancerization. J Biochem Mol Biol 2007; 40: 142–150.

    CAS  PubMed Central  Google Scholar 

  19. Yamashita S, Tsujino Y, Moriguchi K, Tatematsu M, Ushijima T . Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2′-deoxycytidine treatment and oligonucleotide microarray. Cancer Sci 2006; 97: 64–71.

    Article  CAS  Google Scholar 

  20. Ushijima T, Watanabe N, Shimizu K, Miyamoto K, Sugimura T, Kaneda A . Decreased fidelity in replicating CpG methylation patterns in cancer cells. Cancer Res 2005; 65: 11–17.

    CAS  PubMed  Google Scholar 

  21. Ding L, Wang Z, Yan J, Yang X, Liu A, Qiu W et al. Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-beta-like signaling pathway. J Clin Invest 2009; 119: 349–361.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Niu C, Liang C, Guo J, Cheng L, Zhang H, Qin X et al. Downregulation and growth inhibitory role of FHL1 in lung cancer. Int J Cancer 2012; 130: 2549–2556.

    Article  CAS  Google Scholar 

  23. Li X, Jia Z, Shen Y, Ichikawa H, Jarvik J, Nagele RG et al. Coordinate suppression of Sdpr and Fhl1 expression in tumors of the breast, kidney, and prostate. Cancer Sci 2008; 99: 1326–1333.

    Article  CAS  Google Scholar 

  24. Matsumoto M, Kawakami K, Enokida H, Toki K, Matsuda R, Chiyomaru T et al. CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer. Int J Mol Med 2010; 26: 241–247.

    CAS  PubMed  Google Scholar 

  25. Sakashita K, Mimori K, Tanaka F, Kamohara Y, Inoue H, Sawada T et al. Clinical significance of loss of Fhl1 expression in human gastric cancer. Ann Surg Oncol 2008; 15: 2293–2300.

    Article  Google Scholar 

  26. Shen Y, Jia Z, Nagele RG, Ichikawa H, Goldberg GS . SRC uses Cas to suppress Fhl1 in order to promote nonanchored growth and migration of tumor cells. Cancer Res 2006; 66: 1543–1552.

    Article  CAS  Google Scholar 

  27. Enomoto S, Maekita T, Tsukamoto T, Nakajima T, Nakazawa K, Tatematsu M et al. Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci 2007; 98: 1853–1861.

    Article  CAS  Google Scholar 

  28. Ota N, Kawakami K, Okuda T, Takehara A, Hiranuma C, Oyama K et al. Prognostic significance of p16(INK4a) hypermethylation in non-small cell lung cancer is evident by quantitative DNA methylation analysis. Anticancer Res 2006; 26: 3729–3732.

    CAS  PubMed  Google Scholar 

  29. Matsusaka K, Kaneda A, Nagae G, Ushiku T, Kikuchi Y, Hino R et al. Classification of Epstein-Barr virus-positive gastric cancers by definition of DNA methylation epigenotypes. Cancer Res 2011; 71: 7187–7197.

    Article  CAS  Google Scholar 

  30. Ding L, Niu C, Zheng Y, Xiong Z, Liu Y, Lin J et al. FHL1 interacts with oestrogen receptors and regulates breast cancer cell growth. J Cell Mol Med 2011; 15: 72–85.

    Article  CAS  Google Scholar 

  31. Shathasivam T, Kislinger T, Gramolini AO . Genes proteins and complexes: the multifaceted nature of FHL family proteins in diverse tissues. J Cell Mol Med 2010; 14: 2702–2720.

    Article  CAS  PubMed Central  Google Scholar 

  32. Achyut BR, Yang L . Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment. Gastroenterol 2011; 141: 1167–1178.

    Article  CAS  Google Scholar 

  33. Niwa T, Tsukamoto T, Toyoda T, Mori A, Tanaka H, Maekita T et al. Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells. Cancer Res 2010; 70: 1430–1440.

    Article  CAS  Google Scholar 

  34. Panning B, Jaenisch R . RNA and the epigenetic regulation of X chromosome inactivation. Cell 1998; 93: 305–308.

    Article  CAS  Google Scholar 

  35. Moriguchi K, Yamashita S, Tsujino Y, Tatematsu M, Ushijima T . Larger numbers of silenced genes in cancer cell lines with increased de novo methylation of scattered CpG sites. Cancer Lett 2007; 249: 178–187.

    Article  CAS  Google Scholar 

  36. Lauren P . The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965; 64: 31–49.

    Article  CAS  Google Scholar 

  37. Fukayama M, Hayashi Y, Iwasaki Y, Chong J, Ooba T, Takizawa T et al. Epstein-Barr virus-associated gastric carcinoma and Epstein-Barr virus infection of the stomach. Lab Invest 1994; 71: 73–81.

    CAS  PubMed  Google Scholar 

  38. Luo B, Wang Y, Wang XF, Liang H, Yan LP, Huang BH et al. Expression of Epstein-Barr virus genes in EBV-associated gastric carcinomas. World J Gastroenterol 2005; 11: 629–633.

    Article  CAS  PubMed Central  Google Scholar 

  39. Cheng H, Bjerknes M, Amar J . Methods for the determination of epithelial cell kinetic parameters of human colonic epithelium isolated from surgical and biopsy specimens. Gastroenterol 1984; 86: 78–85.

    CAS  Google Scholar 

  40. Kaneda A, Kaminishi M, Sugimura T, Ushijima T . Decreased expression of the seven ARP2/3 complex genes in human gastric cancers. Cancer Lett 2004; 212: 203–210.

    Article  CAS  Google Scholar 

  41. Hosoya K, Yamashita S, Ando T, Nakajima T, Itoh F, Ushijima T . Adenomatous polyposis coli 1A is likely to be methylated as a passenger in human gastric carcinogenesis. Cancer Lett 2009; 285: 182–189.

    Article  CAS  Google Scholar 

  42. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR . Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51–59.

    Article  CAS  Google Scholar 

  43. Liang CC, Park AY, Guan JL . In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329–333.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr Yanagihara and Dr Yasui for their kind gift of cell lines. This study was supported by a Grant-in-Aid for the Third-term Comprehensive Cancer Control Strategy from the Ministry of Health, Labour and Welfare, Japan, and by the National Cancer Center Research and Development Fund. TA is a recipient of the Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to T Ushijima.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Asada, K., Ando, T., Niwa, T. et al. FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect. Oncogene 32, 2140–2149 (2013). https://doi.org/10.1038/onc.2012.228

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.228

Keywords

This article is cited by

Search

Quick links