Skip to main content

Advertisement

SpringerLink
Log in

Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma

  • Original Article—Liver, Pancreas, and Biliary Tract
  • Published:
Journal of Gastroenterology Aims and scope Submit manuscript

Abstract

Background

Genetic and epigenetic alterations are the two key mechanisms in the development of hepatocellular carcinoma (HCC). However, how they contribute to hepatocarcinogenesis and the correlation between them has not been fully elucidated.

Methods

A total of 48 paired HCCs and noncancerous tissues were used to detect loss of heterozygosity (LOH) and the methylation profiles of five tumor suppressor genes (RASSF1A, BLU, FHIT, CRBP1, and HLTF) on chromosome 3 by using polymerase chain reaction (PCR) and methylation-specific PCR. Gene expression was analyzed by immunohistochemistry and reverse transcription (RT)-PCR.

Results

Sixteen of 48 (33.3 %) HCCs had LOH on at least one locus on chromosome 3, and two smallest common deleted regions (3p22.3-24.3 and 3p12.3-14.2) were identified. RASSF1A, BLU, and FHIT showed very high frequencies of methylation in HCCs (100, 81.3, and 64.6 %, respectively) and noncancerous tissues, but not in liver tissues from control patients. Well-differentiated HCCs showed high methylation frequencies of these genes but very low frequencies of LOH. Furthermore, BLU methylation was associated with an increased level of alpha-fetoprotein, and FHIT methylation was inversely correlated with HCC recurrence. In comparison, CRBP1 showed moderate frequencies of methylation, while HLTF showed low frequencies of methylation, and CRBP1 methylation occurred mainly in elderly patients. Treatment with 5-aza-2′-deoxycytidine demethylated at least one of these genes and restored their expression in a DNA methylation-dependent or -independent manner.

Conclusions

Hypermethylation of RASSF1A, BLU, and FHIT is a common and very early event in hepatocarcinogenesis; CRBP1 methylation may also be involved in the later stage. Although LOH was not too frequent on chromosome 3, it may play a role as another mechanism in hepatocarcinogenesis.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Midorikawa Y, Yamamoto S, Ishikawa S, Kamimura N, Igarashi H, Sugimura H, et al. Molecular karyotyping of human hepatocellular carcinoma using single-nucleotide polymorphism arrays. Oncogene. 2006;25:5581–90.

    Article  PubMed  CAS  Google Scholar 

  2. Herath NI, Kew MC, Walsh MD, Young J, Powell LW, Leggett BA, et al. Reciprocal relationship between methylation status and loss of heterozygosity at the p14(ARF) locus in Australian and South African hepatocellular carcinomas. J Gastroenterol Hepatol. 2002;17:301–7.

    Article  PubMed  CAS  Google Scholar 

  3. Pang JZ, Qin LX, Ren N, Hei ZY, Ye QH, Jia WD, et al. Loss of heterozygosity at D8S298 is a predictor for long-term survival of patients with tumor-node-metastasis stage I of hepatocellular carcinoma. Clin Cancer Res. 2007;13:7363–9.

    Article  PubMed  CAS  Google Scholar 

  4. Zhang SH, Cong WM, Xian ZH, Wu MC. Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China. World J Gastroenterol. 2005;11:3034–9.

    PubMed  CAS  Google Scholar 

  5. Zhao X, He M, Wan D, Ye Y, He Y, Han L, et al. The minimum LOH region defined on chromosome 17p13.3 in human hepatocellular carcinoma with gene content analysis. Cancer Lett. 2003;190:221–32.

    Article  PubMed  CAS  Google Scholar 

  6. Shao J, Li Y, Li H, Wu Q, Hou J, Liew C. Deletion of chromosomes 9p and 17 associated with abnormal expression of p53, p16/MTS1 and p15/MTS2 gene protein in hepatocellular carcinomas. Chin Med J (Engl). 2000;113:817–22.

    CAS  Google Scholar 

  7. Midorikawa Y, Yamamoto S, Tsuji S, Kamimura N, Ishikawa S, Igarashi H, et al. Allelic imbalances and homozygous deletion on 8p23.2 for stepwise progression of hepatocarcinogenesis. Hepatology. 2009;49:513–22.

    Article  PubMed  CAS  Google Scholar 

  8. Li SP, Wang HY, Li JQ, Zhang CQ, Feng QS, Huang P, et al. Genome-wide analyses on loss of heterozygosity in hepatocellular carcinoma in Southern China. J Hepatol. 2001;34:840–9.

    Article  PubMed  CAS  Google Scholar 

  9. Riquelme E, Tang M, Baez S, Diaz A, Pruyas M, Wistuba II, et al. Frequent epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in gallbladder carcinoma. Cancer Lett. 2007;250:100–6.

    Article  PubMed  CAS  Google Scholar 

  10. Martin SA, Hewish M, Lord CJ, Ashworth A. Genomic instability and the selection of treatments for cancer. J Pathol. 2010;220:281–9.

    PubMed  CAS  Google Scholar 

  11. Dreijerink K, Braga E, Kuzmin I, Geil L, Duh FM, Angeloni D, et al. The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis. Proc Natl Acad Sci USA. 2001;98:7504–9.

    Article  PubMed  CAS  Google Scholar 

  12. Sharp TV, Al-Attar A, Foxler DE, Ding L, de A Vallim TQ, Zhang Y et al. The chromosome 3p21.3-encoded gene, LIMD1, is a critical tumor suppressor involved in human lung cancer development. Proc Natl Acad Sci USA. 2008;105:19932–7.

  13. Zhang Y, Wang R, Song H, Huang G, Yi J, Zheng Y, et al. Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer. Cancer Lett. 2011;303:21–8.

    Article  PubMed  CAS  Google Scholar 

  14. Liao X, Siu MK, Chan KY, Wong ES, Ngan HY, Chan QK, et al. Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesis. Int J Cancer. 2008;123:296–302.

    Article  PubMed  CAS  Google Scholar 

  15. Hsu HS, Chen TP, Hung CH, Wen CK, Lin RK, Lee HC, et al. Characterization of a multiple epigenetic marker panel for lung cancer detection and risk assessment in plasma. Cancer. 2007;110:2019–26.

    Article  PubMed  CAS  Google Scholar 

  16. Hesson LB, Cooper WN, Latif F. Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene. 2007;26:7283–301.

    Article  PubMed  CAS  Google Scholar 

  17. Chang JW, Hsu HS, Ni HJ, Chuang CT, Hsiung CH, Huang TH, et al. Distinct epigenetic domains separated by a CTCF bound insulator between the tandem genes, BLU and RASSF1A. PLoS ONE. 2010;5:e12847.

    Article  PubMed  Google Scholar 

  18. Hesson L, Bieche I, Krex D, Criniere E, Hoang-Xuan K, Maher ER, et al. Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas. Oncogene. 2004;23:2408–19.

    Article  PubMed  CAS  Google Scholar 

  19. Marsit CJ, Kim DH, Liu M, Hinds PW, Wiencke JK, Nelson HH, et al. Hypermethylation of RASSF1A and BLU tumor suppressor genes in non-small cell lung cancer: implications for tobacco smoking during adolescence. Int J Cancer. 2005;114:219–23.

    Article  PubMed  CAS  Google Scholar 

  20. Agathanggelou A, Dallol A, Zochbauer-Muller S, Morrissey C, Honorio S, Hesson L, et al. Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers. Oncogene. 2003;22:1580–8.

    Article  PubMed  CAS  Google Scholar 

  21. Ki KD, Lee SK, Tong SY, Lee JM, Song DH, Chi SG. Role of 5′-CpG island hypermethylation of the FHIT gene in cervical carcinoma. J Gynecol Oncol. 2008;19:117–22.

    Article  PubMed  CAS  Google Scholar 

  22. Toki K, Enokida H, Kawakami K, Chiyomaru T, Tatarano S, Yoshino H, et al. CpG hypermethylation of cellular retinol-binding protein 1 contributes to cell proliferation and migration in bladder cancer. Int J Oncol. 2010;37:1379–88.

    PubMed  CAS  Google Scholar 

  23. Suzuki M, Shigematsu H, Iizasa T, Hiroshima K, Nakatani Y, Minna JD, et al. Exclusive mutation in epidermal growth factor receptor gene, HER-2, and KRAS, and synchronous methylation of nonsmall cell lung cancer. Cancer. 2006;106:2200–7.

    Article  PubMed  CAS  Google Scholar 

  24. Shutoh M, Oue N, Aung PP, Noguchi T, Kuraoka K, Nakayama H, et al. DNA methylation of genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with DNA methylation. Cancer. 2005;104:1609–19.

    Article  PubMed  CAS  Google Scholar 

  25. Wallner M, Herbst A, Behrens A, Crispin A, Stieber P, Goke B, et al. Methylation of serum DNA is an independent prognostic marker in colorectal cancer. Clin Cancer Res. 2006;12:7347–52.

    Article  PubMed  CAS  Google Scholar 

  26. Liew CT, Li HM, Lo KW, Leow CK, Chan JY, Hin LY, et al. High frequency of p16INK4A gene alterations in hepatocellular carcinoma. Oncogene. 1999;18:789–95.

    Article  PubMed  CAS  Google Scholar 

  27. Zhang C, Li H, Zhou G, Zhang Q, Zhang T, Li J, et al. Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells. J Pathol. 2007;212:134–42.

    Article  PubMed  CAS  Google Scholar 

  28. Liew CT, Li HM, Lo KW, Leow CK, Lau WY, Hin LY, et al. Frequent allelic loss on chromosome 9 in hepatocellular carcinoma. Int J Cancer. 1999;81:319–24.

    Article  PubMed  CAS  Google Scholar 

  29. Zhang C, Li H, Wang Y, Liu W, Zhang Q, Zhang T, et al. Epigenetic inactivation of the tumor suppressor gene RIZ1 in hepatocellular carcinoma involves both DNA methylation and histone modifications. J Hepatol. 2010;53:889–95.

    Article  PubMed  CAS  Google Scholar 

  30. Liu Z, Zhou G, Nakamura M, Koike E, Li Y, Ozaki T, et al. Encapsulated follicular thyroid tumor with equivocal nuclear changes, so-called well-differentiated tumor of uncertain malignant potential: a morphological, immunohistochemical, and molecular appraisal. Cancer Sci. 2011;102:288–94.

    Article  PubMed  CAS  Google Scholar 

  31. Shiraishi K, Okita K, Kusano N, Harada T, Kondoh S, Okita S, et al. A comparison of DNA copy number changes detected by comparative genomic hybridization in malignancies of the liver, biliary tract and pancreas. Oncology. 2001;60:151–61.

    Article  PubMed  CAS  Google Scholar 

  32. Yin DT, Wang L, Sun J, Yin F, Yan Q, Shen R, et al. Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma. Int J Clin Exp Pathol. 2010;3:482–91.

    PubMed  CAS  Google Scholar 

  33. Arafa M, Kridelka F, Mathias V, Vanbellinghen JF, Renard I, Foidart JM, et al. High frequency of RASSF1A and RARb2 gene promoter methylation in morphologically normal endometrium adjacent to endometrioid adenocarcinoma. Histopathology. 2008;53:525–32.

    PubMed  CAS  Google Scholar 

  34. Zhang Q, Ying J, Li J, Fan Y, Poon FF, Ng KM, et al. Aberrant promoter methylation of DLEC1, a critical 3p22 tumor suppressor for renal cell carcinoma, is associated with more advanced tumor stage. J Urol. 2010;184:731–7.

    Article  PubMed  CAS  Google Scholar 

  35. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol. 2003;163:1101–7.

    Article  PubMed  CAS  Google Scholar 

  36. Li B, Liu W, Wang L, Li M, Wang J, Huang L, et al. CpG island methylator phenotype associated with tumor recurrence in tumor-node-metastasis stage I hepatocellular carcinoma. Ann Surg Oncol. 2010;17:1917–26.

    Article  PubMed  Google Scholar 

  37. Miyoshi H, Fujie H, Moriya K, Shintani Y, Tsutsumi T, Makuuchi M, et al. Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma. J Gastroenterol. 2004;39:563–9.

    Article  PubMed  CAS  Google Scholar 

  38. Svrcek M, Buhard O, Colas C, Coulet F, Dumont S, Massaoudi I, et al. Methylation tolerance due to an O6-methylguanine DNA methyltransferase (MGMT) field defect in the colonic mucosa: an initiating step in the development of mismatch repair-deficient colorectal cancers. Gut. 2010;59:1516–26.

    Article  PubMed  CAS  Google Scholar 

  39. Guo M, House MG, Hooker C, Han Y, Heath E, Gabrielson E, et al. Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res. 2004;10:5131–6.

    Article  PubMed  CAS  Google Scholar 

  40. Zhang YJ, Wu HC, Shen J, Ahsan H, Tsai WY, Yang HI, et al. Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res. 2007;13:2378–84.

    Article  PubMed  CAS  Google Scholar 

  41. Kang S, Kim JW, Kang GH, Lee S, Park NH, Song YS, et al. Comparison of DNA hypermethylation patterns in different types of uterine cancer: cervical squamous cell carcinoma, cervical adenocarcinoma and endometrial adenocarcinoma. Int J Cancer. 2006;118:2168–71.

    Article  PubMed  CAS  Google Scholar 

  42. Kim JJ, Chung SW, Kim JH, Kim JW, Oh JS, Kim S, et al. Promoter methylation of helicase-like transcription factor is associated with the early stages of gastric cancer with family history. Ann Oncol. 2006;17:657–62.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Research Grant Committee, Hong Kong (No. CUHK4066/02M-2140319), the National Natural Science Foundation of China (No. 30801108), the Research Fund for the Doctoral Program of Higher Education of China (No. 200804221072), the Foundation for Excellent Young Scientists of Shandong Province (No. BS2009SW036), and the Independent Innovation Foundation of Shandong University (No. IIFSDU-2009TS125).

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Institute of Pathology and Pathophysiology, Shandong University School of Medicine, Jinan, 250012, China

    Xiaoying Zhang, Zhiyan Liu, Gengyin Zhou, Qinghui Zhang, Tingguo Zhang, Jianping Zhang & Cuijuan Zhang

  2. Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, N.T., China

    Hiu Ming Li

Authors
  1. Xiaoying Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiu Ming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhiyan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gengyin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qinghui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tingguo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jianping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cuijuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cuijuan Zhang.

Additional information

X. Zhang and H. M. Li contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 110 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, X., Li, H.M., Liu, Z. et al. Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma. J Gastroenterol 48, 132–143 (2013). https://doi.org/10.1007/s00535-012-0621-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00535-012-0621-0

Keywords

Search

Navigation