Skip to main content
PMC home page
Cancer Science logoLink to Cancer Science
. 2005 Aug 19;95(9):736–740. doi: 10.1111/j.1349-7006.2004.tb03254.x

Promoter hypermethylation of DAP‐kinase is associated with poor survival in primary biliary tract carcinoma patients

Tomohiro Tozawa 1, Gen Tamura 1,✉, Teiichiro Honda 1, Shin‐ichi Nawata 2, Wataru Kimura 2, Naohiko Makino 3, Sumio Kawata 3, Tamotsu Sugai 4, Takayuki Suto 5, Teiichi Motoyama 1
PMCID: PMC11159369  PMID: 15471559

Abstract

To clarify the clinicopathological significance of promoter hypermethylation of tumor suppressor and tumor‐related genes in biliary tract carcinomas, we examined the promoter methylation status of multiple genes in primary biliary tract carcinomas. These consisted of carcinomas of the bile duct, gallbladder, and duodenal ampulla. Surgical specimens were obtained from a total of 37 patients with biliary tract carcinoma. The cohort consisted of 23 patients with bile duct carcinoma, 9 patients with gallbladder carcinoma, and 5 patients with ampullary carcinoma. The methylation status of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 was examined by methylation‐specif ic polymerase chain reaction (MSP). The correlation between methylation status and clinicopathological characteristics was then assessed. The methylation frequencies of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 genes were 16.2%, 21.4%, 27.0%, 8.1%, 24.3%, 27.0%, and 56.8%, respectively, in primary biliary tract carcinomas. The number of methylated genes per sample was 2.17±0.28 (average±SD) in bile duct carcinomas, 1.80±0.97 in ampullary carcinomas, and 0.89±0.35 in gallbladder carcinomas, with a statistically significant difference between bile duct carcinomas and gallbladder carcinomas (P=0.02). As for clinicopathological significance, patients with a methylated RUNX3 promoter were significantly older than those with unmethylated RUNX3 (P=0.01), and DAP‐kinase methylation was more frequent in poorly differentiated tumors than in well to moderately differentiated ones (P=0.04). The overall survival rate was significantly lower in patients with methylated DAP‐kinase (P=0.009) or RUNX3 (P=0.034) compared to those with unmethylated genes. Furthermore, DAP‐kinase methylation‐positive status was independently associated with poor survival in multivariate analyses (hazard ratio=8.71, P=0.024). A significant proportion of primary biliary tract carcinomas exhibited promoter hypermethylation of tumor suppressor and tumor‐related genes, although bile duct carcinomas are more prone to being affected by promoter methylation than are gallbladder carcinomas. Hypermethylation of DAP‐kinase appears to be a significant prognostic factor in primary biliary tract carcinomas.

References

  • 1. Chen MF, Jan YY, Wang CS, Jeng LB, Hwang TL. Clinical experience in 20 hepatic resections for peripheral cholangiocarcinoma. Cancer 1989; 64: 2226–32. [DOI] [PubMed] [Google Scholar]
  • 2. Henson DE, Albores SJ, Corle D. Carcinoma of the gallbladder. Histologic types, stage of disease, grade, and survival rates. Cancer 1992; 70: 1493–7. [DOI] [PubMed] [Google Scholar]
  • 3. Henson DE, Albores SJ, Corle D. Carcinoma of the extrahepatic bile ducts. Histologic types, stage of disease, grade, and survival rates. Cancer 1992; 70: 1498–501. [DOI] [PubMed] [Google Scholar]
  • 4. Kass SU, Pruss D, Wolffe AP. How does DNA methylation repress transcription Trends Genet 1997; 13: 444–9. [DOI] [PubMed] [Google Scholar]
  • 5. Razin A, Cedar H. DNA methylation and gene expression. Microbiol Rev 1991; 55: 451–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999; 2: 163–7. [DOI] [PubMed] [Google Scholar]
  • 7. Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001; 61: 3225–9. [PubMed] [Google Scholar]
  • 8. Kim SG, Chan AO, Wu TT, Issa JP, Hamilton SR, Rashid A. Epigenetic and genetic alternations in duodenal carcinomas are distinct from biliary and ampullary carcinomas. Gastroenterology 2003; 124: 1300–10. [DOI] [PubMed] [Google Scholar]
  • 9. Lee S, Kim WH, Jung HY, Yang MH, Kang GH. Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma. Am J Pathol 2002; 161: 1015–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Wong N, Li L, Tsang K, Lai PB, To KF, Johnson PJ. Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma. J Hepatol 2002; 37: 633–9. [DOI] [PubMed] [Google Scholar]
  • 11. Tannapfel A, Sommerer F, Benicke M, Weinans L, Katalinic A, Geissler F, Uhlmann D, Hauss J, Wittekind C. Genetic and epigenetic alterations of the INK4a‐ARF pathway in cholangiocarcinoma. J Pathol 2002; 197: 624–31. [DOI] [PubMed] [Google Scholar]
  • 12. Tannapfel A, Benicke M, Katalinic A, Uhlmann D, Kockerling F, Hauss J, Wittekind C. Frequency of p16 (INK4A) alterations and K‐ras mutations in intrahepatic cholangiocarcinoma of the liver. Gut 2000; 47: 721–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Caca K, Feisthammel J, Klee K, Tannapfel A, Witzigmann H, Wittekind C, Mossner J, Berr F. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines. Int J Cancer 2002; 97: 481–8. [DOI] [PubMed] [Google Scholar]
  • 14. House MG, Wistuba II, Argani P, Guo M, Schulick RD, Hruban RH, Herman JG, Maitra A. Progression of gene hypermethylation in gallstone disease leading to gallbladder cancer. Ann Surg Oncol 2003; 10: 882–9. [DOI] [PubMed] [Google Scholar]
  • 15. Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature 2000; 406: 430–5. [DOI] [PubMed] [Google Scholar]
  • 16. Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ, Lee KY, Nomura S, Lee CW, Han SB, Kim HM, Kim WJ, Yamamoto H, Yamashita N, Yano T, Ikeda T, Itohara S, Inazawa J, Abe T, Hagiwara A, Yamagishi H, Ooe A, Kaneda A, Sugimura T, Ushijima T, Bae SC, Ito Y Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 2002; 109: 113–24. [DOI] [PubMed] [Google Scholar]
  • 17. Toyota M, Sasaki Y, Satoh A, Ogi K, Kikuchi T, Suzuki H, Mita H, Tanaka N, Itoh F, Issa JP, Jair KW, Schuebel KE, Imai K, Tokino T. Epigenetic inactivation of CHFR in human tumors. Proc Natl Acad Sci USA 2003; 100: 7818–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Corn PG, Summers MK, Fogt F, Virmani AK, Gazdar AF, Halazonetis TD, El‐Deiry WS. Frequent hypermethylation of the 5′ CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non‐small cell lung cancer. Carcinogenesis 2003; 24: 47–51. [DOI] [PubMed] [Google Scholar]
  • 19. Shibata Y, Haruki N, Kuwabara Y, Ishiguro H, Shinoda N, Sato A, Kimura M, Koyama H, Toyama T, Nishiwaki T, Kudo J, Terashita Y, Konishi S, Sugiura H, Fujii Y. Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis 2002; 23: 1695–9. [DOI] [PubMed] [Google Scholar]
  • 20. Mizuno K, Osada H, Konishi H, Tatematsu Y, Yatabe Y, Mitsudomi T, Fujii Y, Takahashi T. Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene 2002; 21: 2328–33. [DOI] [PubMed] [Google Scholar]
  • 21. Waki T, Tamura G, Sato M, Terashima M, Nishizuka S, Motoyama T. Promoter methylation status of DAP‐kinase and RUNX3 genes in neoplastic and non‐neoplastic gastric epithelia. Cancer Sci 2003; 94: 360–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Kato N, Tamura G, Fukase M, Shibuya H, Motoyama T. Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants. Am J Pathol 2003; 163: 387–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Waki T, Tamura G, Tsuchiya T, Sato K, Nishizuka S, Motoyama T. Promoter methylation status of E‐cadherin, hMLH1, and p16 genes in nonneoplastic gastric epithelia. Am. J Pathol 2002; 161: 399–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Fleisher AS, Esteller M, Wang S, Tamura G, Suzuki H, Yin J, Zou TT, Abraham JM, Kong D, Smolinski KN, Shi YQ, Rhyu MG, Powell SM, James SP, Wilson KT, Herman JG, Meltzer SJ. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res 1999; 59: 1090–5. [PubMed] [Google Scholar]
  • 25. Lo KW, Kwong J, Hui AB, Chan SY, To KF, Chan AS, Chow LS, Teo PM, Johnson PJ, Huang DP. High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma. Cancer Res 2001; 61: 3877–81. [PubMed] [Google Scholar]
  • 26. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation‐specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. 1 Hypermethylation of the DAP‐kinase CpG island is a common alteration in B‐cell malignancies. Blood 1999; 93: 4347–53. [PubMed] [Google Scholar]
  • 28. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell‐cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993; 366: 704–7. [DOI] [PubMed] [Google Scholar]
  • 29. Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, Kane M, Earabino C, Lipford J, Lindblom A, Tannergård P, Bollag RJ, Godwin AR, Ward DC, Nordenskjld M, Fishel R, Kolodner R, Liskay RM. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non‐polyposis colon cancer. Nature 1994; 368: 258–61. [DOI] [PubMed] [Google Scholar]
  • 30. Inbal B, Cohen O, Polak‐Charcon S, Kopolovic J, Vadai E, Eisenbach L, Kimchi A. DAP kinase links the control of apoptosis to metastasis. Nature 1997; 390: 180–4. [DOI] [PubMed] [Google Scholar]
  • 31. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991; 251: 1451–5. [DOI] [PubMed] [Google Scholar]
  • 32. Kang GH, Lee S, Kim JS, Jung HY Profile of aberrant CpG island methylation along the multistep pathway of gastric Carcinogenesis. Lab Invest 2003; 83: 635–41. [DOI] [PubMed] [Google Scholar]
  • 33. Yanagawa N, Tamura G, Oizumi H, Takahashi N, Shimazaki Y, Motoyama T. Promoter hypermethylation of tumor suppressor and tumor‐related genes in non‐small cell lung cancers. Cancer Sci 2003; 94: 589–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet 2000; 25: 315–9. [DOI] [PubMed] [Google Scholar]
  • 35. Dammann R, Yang G, Pfeifer GP. Hypermethylation of the CpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res 2001; 61: 3105–9. [PubMed] [Google Scholar]
  • 36. 1 Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene 2001; 20: 1509–18. [DOI] [PubMed] [Google Scholar]
  • 37. Dreijerink K, Braga E, Kuzmin I, Geil L, Duh FM, Angeloni D, Zbar B, Lerman MI, Stanbridge EJ, Minna JD, Protopopov A, Li J, Kashuba V, Klein G, Zabarovsky ER. 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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Maruyama R, Toyooka S, Toyooka KO, Virmani AK, Zochbauer‐Muller S, Farinas AJ, Minna JD, McConnell J, Frenkel EP, Gazdar AF. Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clin Cancer Res 2002; 8: 514–9. [PubMed] [Google Scholar]
  • 39. Schagdarsurengin U, Gimm O, Hoang‐Vu C, Dralle H, Pfeifer GP, Dammann R. Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma. Cancer Res 2002; 62: 3698–701. [PubMed] [Google Scholar]
  • 40. Byun DS, Lee MG, Chae KS, Ryu BG, Chi SG. Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma. Cancer Res 2001; 61: 7034–8. [PubMed] [Google Scholar]
  • 41. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998; 396: 643–9. [DOI] [PubMed] [Google Scholar]
  • 42. Suto T, Sasaki K, Sugai T, Kanno S, Saito K. Heterogeneity in the nuclear DNA content of cells in carcinomas of the biliary tract and pancreas. Cancer 1993; 72: 2920–8. [DOI] [PubMed] [Google Scholar]
  • 43. Waki T, Tamura G, Sato M, Motoyama T. Age‐related methylation of tumor suppressor and tumor‐related genes: an analysis of autopsy samples. Oncogene 2003; 22: 4128–33. [DOI] [PubMed] [Google Scholar]
  • 44. Tsuchiya T, Tamura G, Sato K, Endoh Y, Sakata K, Jin Z, Motoyama T, Usuba O, Kimura W, Nishizuka S, Wilson KT, James SP, Yin J, Fleisher AS, Zou T, Silverberg SG, Kong D, Meltzer SJ. Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia. Oncogene 2000; 19: 3642–6. [DOI] [PubMed] [Google Scholar]
  • 45. Virmani AK, Rathi A, Sathyanarayana UG, Padar A, Huang CX, Cunnigham HT, Farinas AJ, Milchgrub S, Euhus DM, Gilcrease M, Herman J, Minna JD, Gazdar AF. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas. Clin Cancer Res 2001; 7: 1998–2004. [PubMed] [Google Scholar]
  • 46. To KF, Leung WK, Lee TL, Yu J, Tong JH, Chan MW, Ng EK, Chung SC, Sung JJ. Promoter hypermethylation of tumor‐related genes in gastric intestinal metaplasia of patients with and without gastric cancer. Int J Cancer 2002; 102: 623–8. [DOI] [PubMed] [Google Scholar]
  • 47. Nakagawa H, Nuovo GJ, Zervos EE, Martin EW Jr, Salovaara R, Aaltonen LA, de la Chapelle A. Age‐related hypermethylation of the 5′ region of MLH1 in normal colonic mucosa is associated with microsatellite‐unstable colorectal cancer development. Cancer Res 2001; 61: 6991–5. [PubMed] [Google Scholar]
  • 48. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age‐related CpG island methylation in ulcerative colitis. Cancer Res 2001; 61: 3573–7. [PubMed] [Google Scholar]
  • 49. Yanagawa N, Tamura G, Oizumi H, Takahashi N, Shimazaki Y, Motoyama T. Frequent epigenetic silencing of the p16 gene in non‐small cell lung cancers of tobacco smokers. Jpn J Cancer Res 2002; 93: 1107–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Chan AO, Lam SK, Wong BC, Wong WM, Yuen MF, Yeung YH, Hui WM, Rashid A, Kwong YL. Promoter methylation of E‐cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut 2003; 52: 502–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Matsumoto H, Nagao M, Ogawa S, Kanehiro H, Hisanaga M, Ko S, Ikeda N, Fujii H, Koyama F, Mukogawa T, Nakajima Y. Prognostic significance of death‐associated protein‐kinase expression in hepatocellular carcinomas. Anticancer Res 2003; 23: 1333–41. [PubMed] [Google Scholar]
  • 52. Kim DH, Nelson HH, Wiencke JK, Christiani DC, Wain JC, Mark EJ, Kelsey KT. Promoter methylation of DAP‐kinase: association with advanced stage in non‐small cell lung cancer. Oncogene 2001; 20: 1765–70. [DOI] [PubMed] [Google Scholar]
  • 53. Ng MH, To KW, Lo KW, Chan S, Tsang KS, Cheng SH, Ng HK. Frequent death‐associated protein kinase promoter hypermethylation in multiple myeloma. Clin Cancer Res 2001; 7: 1724–9. [PubMed] [Google Scholar]
  • 54. Abe M, Okochi E, Kuramoto T, Kaneda A, Takato T, Sugimura T, Ushijima T. Cloning of the 5′ upstream region of the rat p16 gene and its role in silencing. Jpn J Cancer Res 2002; 93: 1100–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cancer Science are provided here courtesy of Wiley

RESOURCES