J Korean Med Sci.  2012 Jun;27(6):594-604. 10.3346/jkms.2012.27.6.594.

Pharmacological Unmasking Microarray Approach-Based Discovery of Novel DNA Methylation Markers for Hepatocellular Carcinoma

Affiliations
  • 1Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea. ghkang@snu.ac.kr
  • 2Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
  • 3Department of General Surgery, Seoul National University College of Medicine, Seoul, Korea.
  • 4Department of Pathology, Korea University School of Medicine, Seoul, Korea.

Abstract

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

Keyword

CpG Islands; DNA Methylation; Carcinoma, Hepatocellular; Microarray; Prognosis

MeSH Terms

Azacitidine/analogs & derivatives/pharmacology
Carcinoma, Hepatocellular/*genetics/mortality
Cell Line, Tumor
CpG Islands
DNA Methylation/*drug effects
Down-Regulation
Female
Hep G2 Cells
Humans
Hydroxamic Acids/pharmacology
Liver/metabolism
Liver Neoplasms/*genetics/mortality
Male
Middle Aged
Oligonucleotide Array Sequence Analysis
Promoter Regions, Genetic
Survival Analysis
Tumor Markers, Biological/*genetics
Hydroxamic Acids
Tumor Markers, Biological
Azacitidine

Figure

  • Fig. 1 Flow chart for selection of candidate genes. Screening of candidate tumor suppressor genes (TSGs) was performed in 5 hepatocellular carcinoma (HCC) cell lines treated with 5 µM 5-aza-2'-deoxycytidine (AZA) or 300 nM trichostatin A (TSA) by using a 24,526-oligonucleotide mRNA microarray. We obtained 793 candidates whose gene expression did not increase with TSA treatment (< 1.4-fold) but increased more than 2-fold after AZA treatment. We excluded genes that do not harbor CpG islands in their promoters or whose methylation status in HCC tumors had already been reported in the literature. We further excluded genes for which adequate oligonucleotide primers could not be designed by using the MSPprimer or MethPrimer software programs. As a result, we selected 380 genes to be examined for their methylation status in HCC cell lines by using methylation-specific PCR (MSP).

  • Fig. 2 Hypermethylation-dependent expression changes. Gene expression changes for the indicated cells treated with trichostatin A (TSA) (x-axis) or 5-aza-2'-deoxycytidine (AZA) (y-axis) are plotted by log-fold change, and individual genes are shown in circles.

  • Fig. 3 Methylation-specific PCR (MSP) analysis of 380 selected genes in 8 hepatocellular carcinoma (HCC) cell lines. The methylated and unmethylated status is indicated by a gray and a white box, respectively.

  • Fig. 4 Frequency of H3K27me3 modification and occupancy rate of SUZ12 and EED in methylated (n = 239) and unmethylated genes (n = 141) in human embryonic stem cells. The chi-squared test was conducted to analyze the significance of the association.

  • Fig. 5 Comparison of ALU and LINE-1 repeats between methylated and unmethylated genes. For ALU counting, the promoter sequence of a specific gene was divided into 20 bins of 1-kb sequence each (10 bins upstream and 10 bins downstream of each gene transcription start site), and the presence of ALU was annotated for each bin. We counted bins containing ALU within a 1-kb sequence. For LINE-1 counting, the promoter sequence of a specific gene was divided into 7 bins of 1-kb sequence each (2 bins upstream and 5 bins downstream of each gene transcription site), and the presence of LINE-1 was annotated for each bin. Bins containing LINE-1 within a 1-kb sequence were counted. Student's t-test was performed to determine the statistical significance of the difference of means between 2 groups.

  • Fig. 6 Methylation-specific PCR (MSP) analysis of 239 genes in 10 pairs of tumor and surrounding nontumor tissue. The methylated and unmethylated status is indicated by a gray and a white box, respectively. Genes were divided into subgroups based on the methylation pattern in hepatocellular carcinoma (HCC) tumors and surrounding nonneoplastic liver tissues (see main text).

  • Fig. 7 Effect of 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA) on gene expression. RNA was isolated from hepatocellular carcinoma (HCC) cell lines left untreated or treated with AZA, TSA, or a combination of AZA and TSA. mRNA was reverse-transcribed, and gene expression was quantitated by real-time PCR and normalized to GAPDH expression.

  • Fig. 8 Methylation frequencies of 33 DNA methylation markers in HCC cell lines (n = 8) and tissue samples (10 paired HCC and non-neoplastic liver tissue samples). Methylation frequencies of these DNA methylation markers in HCC tissue samples were higher than those of non-neoplastic liver tissue samples: the differences were 20% or more.

  • Fig. 9 Methylation frequencies of 33 DNA methylation markers in hepatocellular carcinoma (HCC) tissue samples (n = 95). DNA methylation markers were distributed along the x-axis according to the decreasing order of methylation frequency.

  • Fig. 10 Kaplan-Meier survival curves of 90 hepatocellular carcinoma (HCC) patients. Correlation of (A) HIST1H2AE, and (B) EFEMP2 methylation status with overall survival.


Reference

1. Saxonov S, Berg P, Brutlag DL. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A. 2006. 103:1412–1417.
2. Weber M, Hellmann I, Stadler MB, Ramos L, Pääbo S, Rebhan M, Schübeler D. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet. 2007. 39:457–466.
3. Schuebel KE, Chen W, Cope L, Glöckner SC, Suzuki H, Yi JM, Chan TA, Van Neste L, Van Criekinge W, van den Bosch S, et al. Comparing the DNA hypermethylome with gene mutations in human colorectal cancer. PLoS Genet. 2007. 3:1709–1723.
4. Wood LD, Parsons DW, Jones S, Lin J, Sjóblom T, Leary RJ, Shen D, Boca SM, Barber T, Ptak J, et al. The genomic landscapes of human breast and colorectal cancers. Science. 2007. 318:1108–1113.
5. Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer. 2003. 3:253–266.
6. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M, Arii K, Kaneda A, Tsukamoto T, Tatematsu 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.
7. deVos T, Tetzner R, Model F, Weiss G, Schuster M, Distler J, Steiger KV, Grützmann R, Pilarsky C, Habermann JK, et al. Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer. Clin Chem. 2009. 55:1337–1346.
8. Tänzer M, Balluff B, Distler J, Hale K, Leodolter A, Röcken C, Molnar B, Schmid R, Lofton-Day C, Schuster T, et al. Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions. PLoS One. 2010. 5:e9061.
9. Herbst A, Rahmig K, Stieber P, Philipp A, Jung A, Ofner A, Crispin A, Neumann J, Lamerz R, Kolligs FT. Methylation of NEUROG1 in serum is a sensitive marker for the detection of early colorectal cancer. Am J Gastroenterol. 2011. 106:1110–1118.
10. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol. 2001. 2:533–543.
11. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology. 2007. 132:2557–2576.
12. Chang MH, You SL, Chen CJ, Liu CJ, Lee CM, Lin SM, Chu HC, Wu TC, Yang SS, Kuo HS, et al. Decreased incidence of hepatocellular carcinoma in hepatitis B vaccinees: a 20-year follow-up study. J Natl Cancer Inst. 2009. 101:1348–1355.
13. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009. 59:225–249.
14. Herath NI, Leggett BA, MacDonald GA. Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroenterol Hepatol. 2006. 21:15–21.
15. Villanueva A, Newell P, Chiang DY, Friedman SL, Llovet JM. Genomics and signaling pathways in hepatocellular carcinoma. Semin Liver Dis. 2007. 27:55–76.
16. Kanai Y, Ushijima S, Hui AM, Ochiai A, Tsuda H, Sakamoto M, Hirohashi S. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int J Cancer. 1997. 71:355–359.
17. Calvisi DF, Ladu S, Gorden A, Farina M, Lee JS, Conner EA, Schroeder I, Factor VM, Thorgeirsson SS. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest. 2007. 117:2713–2722.
18. Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A. Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology. 2008. 47:908–918.
19. Shin SH, Kim BH, Jang JJ, Suh KS, Kang GH. Identification of novel methylation markers in hepatocellular carcinoma using a methylation array. J Korean Med Sci. 2010. 25:1152–1159.
20. Yuan Y, Wang J, Li J, Wang L, Li M, Yang Z, Zhang C, Dai JL. Frequent epigenetic inactivation of spleen tyrosine kinase gene in human hepatocellular carcinoma. Clin Cancer Res. 2006. 12:6687–6695.
21. Lu B, Ma Y, Wu G, Tong X, Guo H, Liang A, Cong W, Liu C, Wang H, Wu M, et al. Methylation of Tip30 promoter is associated with poor prognosis in human hepatocellular carcinoma. Clin Cancer Res. 2008. 14:7405–7412.
22. Lee HS, Kim BH, Cho NY, Yoo EJ, Choi M, Shin SH, Jang JJ, Suh KS, Kim YS, Kang GH. Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma. Clin Cancer Res. 2009. 15:812–820.
23. Zhang W, Zhou L, Ding SM, Xie HY, Xu X, Wu J, Chen QX, Zhang F, Wei BJ, Eldin AT, et al. Aberrant methylation of the CADM1 promoter is associated with poor prognosis in hepatocellular carcinoma treated with liver transplantation. Oncol Rep. 2011. 25:1053–1062.
24. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996. 93:9821–9826.
25. Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, Herman JG, Baylin SB. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet. 2002. 31:141–149.
26. Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002. 21:5483–5495.
27. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K, et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell. 2006. 125:301–313.
28. Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, Cui H, Gabo K, Rongione M, Webster M, et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet. 2009. 41:178–186.
29. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, Eden E, Yakhini Z, Ben-Shushan E, Reubinoff BE, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet. 2007. 39:232–236.
30. Estécio MR, Gallegos J, Vallot C, Castoro RJ, Chung W, Maegawa S, Oki Y, Kondo Y, Jelinek J, Shen L, et al. Genome architecture marked by retrotransposons modulates predisposition to DNA methylation in cancer. Genome Res. 2010. 20:1369–1382.
31. Hereford LM, Osley MA, Ludwig TR 2nd, McLaughlin CS. Cell-cycle regulation of yeast histone mRNA. Cell. 1981. 24:367–375.
32. Marzluff WF, Duronio RJ. Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences. Curr Opin Cell Biol. 2002. 14:692–699.
33. Nishida H, Suzuki T, Tomaru Y, Hayashizaki Y. A novel replication-independent histone H2a gene in mouse. BMC Genet. 2005. 6:10.
34. Albig W, Doenecke D. The human histone gene cluster at the D6S105 locus. Hum Genet. 1997. 101:284–294.
35. Albig W, Kioschis P, Poustka A, Meergans K, Doenecke D. Human histone gene organization: nonregular arrangement within a large cluster. Genomics. 1997. 40:314–322.
36. Nishida H, Suzuki T, Ookawa H, Tomaru Y, Hayashizaki Y. Comparative analysis of expression of histone H2a genes in mouse. BMC Genomics. 2005. 6:108.
37. Chu M, Tsuda T. Fibulins in development and heritable disease. Birth Defects Res C Embryo Today. 2004. 72:25–36.
38. de Vega S, Iwamoto T, Yamada Y. Fibulins: multiple roles in matrix structures and tissue functions. Cell Mol Life Sci. 2009. 66:1890–1902.
39. Hucthagowder V, Sausgruber N, Kim KH, Angle B, Marmorstein LY, Urban Z. Fibulin-4: a novel gene for an autosomal recessive cutis laxa syndrome. Am J Hum Genet. 2006. 78:1075–1080.
40. Furuta J, Nobeyama Y, Umebayashi Y, Otsuka F, Kikuchi K, Ushijima T. Silencing of Peroxiredoxin 2 and aberrant methylation of 33 CpG islands in putative promoter regions in human malignant melanomas. Cancer Res. 2006. 66:6080–6086.
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