Skip to main content
SpringerLink
Log in

Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

ABSTRACT

Background

We previously demonstrated in an immunohistochemical study that reduced expression of O6−methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.

Methods

We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.

Results

Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, β-catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.

Conclusions

Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.

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

Similar content being viewed by others

References

  1. T Okusaka (2002) ArticleTitleChemotherapy for biliary tract cancer in Japan Semin Oncol 29 51–3 Occurrence Handle10.1053/sonc.2002.31523 Occurrence Handle1:CAS:528:DC%2BD3sXhslehtLo%3D Occurrence Handle12577234

    Article  CAS  PubMed  Google Scholar 

  2. Y Nimura (2002) ArticleTitleExtended surgery in bilio-pancreatic cancer: the Japanese experience Semin Oncol 29 17–22 Occurrence Handle10.1053/sonc.2002.37371 Occurrence Handle12577229

    Article  PubMed  Google Scholar 

  3. H Paimela A Karppinen K Hockerstedt V Perhoniemi E Kivilaakso (1997) ArticleTitlePoor prognosis of gallbladder cancer persist regardless of improved diagnostic methods. Incidence and results of surgery during 20 years in Helsinki Ann Chir Gynaecol 86 13–17 Occurrence Handle1:STN:280:ByiA38rhsVA%3D Occurrence Handle9181213

    CAS  PubMed  Google Scholar 

  4. A Rashid (2002) ArticleTitleCellular and molecular biology of biliary tract cancers Surg Oncol Clin North Am 11 995–1009 Occurrence Handle10.1016/S1055-3207(02)00042-X

    Article  Google Scholar 

  5. A Loveless (1969) ArticleTitlePossible relevance of O-6 alkylation of deoxyguanosine to the mutagency and carcinogencity of nitrosamines and nitrosamides Nature 13 206–7 Occurrence Handle10.1038/223206a0

    Article  Google Scholar 

  6. AE Pegg (1990) ArticleTitleMammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents Cancer Res 50 6119–29 Occurrence Handle1:CAS:528:DyaK3cXmtVOisLs%3D Occurrence Handle2205376

    CAS  PubMed  Google Scholar 

  7. B Demple A Jacobsson M Olsson P Robins T Lindahl (1982) ArticleTitleRepair of alkylated DNA in Escherichia coli: physical properties of O6−methylguanine-DNA methyltransferase J Biol Chem 257 13776–80 Occurrence Handle1:CAS:528:DyaL38XlvFCqtbs%3D Occurrence Handle6754717

    CAS  PubMed  Google Scholar 

  8. K Sakumi A Shiraishi S Shimizu T Tsuzuki T Ishikawa M Sekiguchi (1997) ArticleTitleMethylnitrosourea-induced tumorigenesis in MGMT gene knockout mice Cancer Res 15 2415–8

    Google Scholar 

  9. SL Gerson JE Trey K Miller NA Berger (1986) ArticleTitleComparison of O6−alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues Carcinogenesis 7 745–9 Occurrence Handle1:CAS:528:DyaL28Xmt1Slurk%3D Occurrence Handle3698202 Occurrence Handle10.1093/carcin/7.5.745

    Article  CAS  PubMed  Google Scholar 

  10. N Kohya K Miyazaki S Matsukura et al. (2002) ArticleTitleDeficient expression of O6−methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and MSH2 is related to poor prognosis in human biliary tract carcinoma Ann Surg Oncol 9 371–9 Occurrence Handle10.1245/aso.2002.9.4.371 Occurrence Handle11986189

    Article  PubMed  Google Scholar 

  11. M Esteller (2003) ArticleTitleRelevance of DNA methylation in the management of cancer Lancet Oncol 4 351–8 Occurrence Handle10.1016/S1470-2045(03)01115-X Occurrence Handle1:CAS:528:DC%2BD3sXkt1OksL0%3D Occurrence Handle12788407

    Article  CAS  PubMed  Google Scholar 

  12. K Hanada M Itoh K Fujii A Tsuchida H Ooishi G Kajiyama (1996) ArticleTitleK-ras and p53 mutations in stag I gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct Cancer 77 452–8 Occurrence Handle10.1002/(SICI)1097-0142(19960201)77:3<452::AID-CNCR5>3.0.CO;2-M Occurrence Handle1:CAS:528:DyaK28Xht12jsL4%3D Occurrence Handle8630951

    Article  CAS  PubMed  Google Scholar 

  13. T Itoi H Watanabe Y Ajioka et al. (1996) ArticleTitleAPC, K-ras codon 12 mutations and p53 gene expression in carcinoma and adenoma of the gallbladder suggest two genetic pathways in gallbladder carcinogenesis Pathol Int 46 333–40 Occurrence Handle1:CAS:528:DyaK28XltVajtb4%3D Occurrence Handle8809879 Occurrence Handle10.1111/j.1440-1827.1996.tb03618.x

    Article  CAS  PubMed  Google Scholar 

  14. S Yoshida T Todoroki Y Ichikawa et al. (1995) ArticleTitleMutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers Cancer Res 55 2756–60 Occurrence Handle1:CAS:528:DyaK2MXmsFyksrk%3D Occurrence Handle7796400

    CAS  PubMed  Google Scholar 

  15. H Watanabe K Date T Itoi et al. (1999) ArticleTitleHistological and genetic changes in malignant transformation of gallbladder adenoma Ann Oncol 10 136–9 Occurrence Handle10.1023/A:1008330012536 Occurrence Handle10436806

    Article  PubMed  Google Scholar 

  16. II Wistuba J Albores-Saavedra (1999) ArticleTitleGenetic abnormalities involved in the pathogeneisis of gallbladder carcinoma J Hepatobiliary Pancreat Surg 6 237–44 Occurrence Handle10.1007/s005340050113 Occurrence Handle1:STN:280:DyaK1Mvlsleiuw%3D%3D Occurrence Handle10526058

    Article  CAS  PubMed  Google Scholar 

  17. N Kohya Y Kitajima K Kitahara K Miyazaki (2003) ArticleTitleMutation analysis of K-ras and β-catenin genes related to O6-methylguanine-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma Int J Mol Med 11 65–9 Occurrence Handle1:CAS:528:DC%2BD3sXpslei Occurrence Handle12469220

    CAS  PubMed  Google Scholar 

  18. SB Baylin JG Herman (2000) ArticleTitleDNA hypermethylation in tumorigenesis: epigenetics joins genetics Trends Genet 16 168–74 Occurrence Handle10.1016/S0168-9525(99)01971-X Occurrence Handle1:CAS:528:DC%2BD3cXit1Sqsbg%3D Occurrence Handle10729832

    Article  CAS  PubMed  Google Scholar 

  19. S Matsukura H Soejima T Nakagawachi et al. (2003) ArticleTitleCpG methylation of MGMT and MLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection Bm J Cancer 88 521–9 Occurrence Handle10.1038/sj.bjc.6600743 Occurrence Handle1:CAS:528:DC%2BD3sXht1Glur0%3D

    Article  CAS  Google Scholar 

  20. M Esteller SR Hamilton PC Burger SB Baylin JG Herman (1999) ArticleTitleInactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia Cancer Res 59 793–7 Occurrence Handle1:CAS:528:DyaK1MXht1Sjurk%3D Occurrence Handle10029064

    CAS  PubMed  Google Scholar 

  21. M Esteller M Sanchez-Cespedes R Rosell D Sidransky SB Baylin JG Herman (1999) ArticleTitleDetection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients Cancer Res 59 67–70 Occurrence Handle1:CAS:528:DyaK1MXmsFOhug%3D%3D Occurrence Handle9892187

    CAS  PubMed  Google Scholar 

  22. J Brabender H Usadel R Metzger et al. (2003) ArticleTitleQuantitative O6-methylguanine-DNA methyltransferase analysis in curatively resected non-small cell lung cancer; association with clinical outcome Clin Cancer Res 9 223–7 Occurrence Handle1:CAS:528:DC%2BD3sXnt1OksQ%3D%3D Occurrence Handle12538473

    CAS  PubMed  Google Scholar 

  23. DJ Wong MT Barrett R Stoger MJ Emond BJ Reid (1997) ArticleTitlep16/INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas Cancer Res 57 2619–22 Occurrence Handle1:CAS:528:DyaK2sXkt12qtro%3D Occurrence Handle9205067

    CAS  PubMed  Google Scholar 

  24. SW Tsao Y Liu X Wang et al. (2003) ArticleTitleThe association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cells Eur J Cancer 39 524–31 Occurrence Handle10.1016/S0959-8049(02)00494-X Occurrence Handle1:CAS:528:DC%2BD3sXhtVemurs%3D Occurrence Handle12751385

    Article  CAS  PubMed  Google Scholar 

  25. AS Fleisher M Esteller S Wang et al. (1999) ArticleTitleHypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability Cancer Res 59 1090–5 Occurrence Handle1:CAS:528:DyaK1MXhslelsLw%3D Occurrence Handle10070967

    CAS  PubMed  Google Scholar 

  26. X Tang FR Khuri JJ Lee et al. (2000) ArticleTitleHypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small cell lung cancer J Natl Cancer Inst 92 1511–6 Occurrence Handle10.1093/jnci/92.18.1511 Occurrence Handle1:CAS:528:DC%2BD3cXntFKqsr0%3D Occurrence Handle10995806

    Article  CAS  PubMed  Google Scholar 

  27. M Toyota N Ahuja M Ohe-Toyota JG Herman SB Baylin J-PJ Issa (1999) ArticleTitleCpG island methylator phenotype in colorectal cancer Proc Natl Acad Sci 96 8681–6 Occurrence Handle10.1073/pnas.96.15.8681 Occurrence Handle1:CAS:528:DyaK1MXkslOktLg%3D Occurrence Handle10411935

    Article  CAS  PubMed  Google Scholar 

  28. M Toyota M Ohe-Toyota N Ahuja J-PJ Issa (2000) ArticleTitleDistinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype Proc Natl Acad Sci USA 97 710–6 Occurrence Handle10.1073/pnas.97.2.710 Occurrence Handle1:CAS:528:DC%2BD3cXot1ahuw%3D%3D Occurrence Handle10639144

    Article  CAS  PubMed  Google Scholar 

  29. MV Brock M Gou Y Akiyama et al. (2003) ArticleTitlePrognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinoma Clin Cancer Res 9 2912–9 Occurrence Handle1:CAS:528:DC%2BD3sXmtF2iu7o%3D Occurrence Handle12912936

    CAS  PubMed  Google Scholar 

  30. MG House JG Herman MZ Guo et al. (2003) ArticleTitleAberrant hypermethylation of tumor suppressor genes in pancreatic endocrine neoplasms Ann Surg Oncol 238 423–32

    Google Scholar 

  31. MG House II Wistuba P Argani et al. (2003) ArticleTitleProgression of gene hypermethylation in gallstone disease leading to gallbladder cancer Ann Surg Oncol 10 882–9 Occurrence Handle10.1245/ASO.2003.02.014 Occurrence Handle14527906

    Article  PubMed  Google Scholar 

  32. S Lee WH Kim H-Y Jung MH Yang GH Kang (2002) ArticleTitleAberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma Am J Patho 161 1015–22 Occurrence Handle1:CAS:528:DC%2BD38XnsFOis74%3D

    CAS  Google Scholar 

  33. JG Herman R Graff S Myohanen BD Nelkin SB Baylin (1996) ArticleTitleMethylation-specific PCR: a novel PCR assay for methylation status of CpG islands Proc Natl Acad Sci USA 93 9821–6 Occurrence Handle1:CAS:528:DyaK28XlsFeht7Y%3D Occurrence Handle8790415 Occurrence Handle10.1073/pnas.93.18.9821

    Article  CAS  PubMed  Google Scholar 

  34. R Paulin GW Grigg MW Davey AA Piper (1998) ArticleTitleUrea improves efficiency of bisulfite-mediated sequencing of 5′-methylcytosine in genomic DNA Nucleric Acids Res 26 5009–10 Occurrence Handle10.1093/nar/26.21.5009 Occurrence Handle1:CAS:528:DyaK1cXnsVKgur0%3D

    Article  CAS  Google Scholar 

  35. A Scarpa P Capelli A Villaneuva et al. (1994) ArticleTitlePancreatic cancer in Europe: Ki-ras gene mutation pattern shows geographical differences Int J Cancer 57 167–71 Occurrence Handle1:CAS:528:DyaK2MXhsFyksQ%3D%3D Occurrence Handle8157353 Occurrence Handle10.1002/ijc.2910570206

    Article  CAS  PubMed  Google Scholar 

  36. A Scarpa P Capelli K Mukai et al. (1993) ArticleTitlePancreatic adenocarcinomas frequently show p53 mutations Am J Pathol 142 1534–43 Occurrence Handle8494051

    PubMed  Google Scholar 

  37. SY Zhang AJ Klein-Szanto ER Sauter et al. (1994) ArticleTitleHigher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck Cancer Res 54 5050–3 Occurrence Handle1:CAS:528:DyaK2cXmt1emu7k%3D Occurrence Handle7923115

    CAS  PubMed  Google Scholar 

  38. S Matsukura K Miyazaki H Yakushiji et al. (2001) ArticleTitleExpression and prognostic significance of O6-methylguanine-DNA methyltransferase in hepatocellular, gastric, and breast cancers Ann Surg Oncol 8 807–16 Occurrence Handle1:STN:280:DC%2BD38%2FkvVyntw%3D%3D Occurrence Handle11776495 Occurrence Handle10.1007/s10434-001-0807-9

    Article  CAS  PubMed  Google Scholar 

  39. Y Kitajima K Miyazaki S Matsukura M Tanaka M. Sekiguchi (2003) ArticleTitleLoss of expression of DNA repair enzymes MGMT, hMLH1, and MSH2 during tumor progression in gastric cancer Gastric Cancer 6 86–95 Occurrence Handle1:CAS:528:DC%2BD2cXntVymt7k%3D Occurrence Handle12861399

    CAS  PubMed  Google Scholar 

  40. M Esteller J Garcia-Foncillas E Andion et al. (2000) ArticleTitleInactivation of the DNA repair gene MGMT and clinical response of gliomas to alkylating agents N Engl J Med 343 1350–4 Occurrence Handle10.1056/NEJM200011093431901 Occurrence Handle1:CAS:528:DC%2BD3cXosFOltLg%3D Occurrence Handle11070098

    Article  CAS  PubMed  Google Scholar 

  41. M Esteller G Gaidano SN Goodman et al. (2002) ArticleTitleHypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma J Natl Cancer Inst 94 26–32 Occurrence Handle1:CAS:528:DC%2BD38Xit1amt7Y%3D Occurrence Handle11773279

    CAS  PubMed  Google Scholar 

  42. M Esteller M Toyota M Sanchez-Cespedes et al. (2000) ArticleTitleInactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis Cancer Res 60 2368–71 Occurrence Handle1:CAS:528:DC%2BD3cXjtV2qtro%3D Occurrence Handle10811111

    CAS  PubMed  Google Scholar 

  43. M Esteller R-A Risques M Toyota et al. (2001) ArticleTitlePromoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutation in p53 in human colorectal tumorigenesis Cancer Res 61 4689–92 Occurrence Handle1:CAS:528:DC%2BD3MXks1GrtLg%3D Occurrence Handle11406538

    CAS  PubMed  Google Scholar 

  44. P Wolf YC Hu K Doffek D Sidransky SA Ahrendt (2001) ArticleTitleO6-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancer Cancer Res 61 8112–7

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Surgery, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga, 849-8501, Japan

    Yasuo Koga MD, Yoshihiko Kitajima MD, PhD, Atsushi Miyoshi MD, Ken Sato MD, Kenji Kitahara MD, PhD & Kohji Miyazaki MD, PhD

  2. Department of Biomolecular Sciences, Division of Molecular Biology & Genetics, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga, 849-8501, Japan

    Hidenobu Soejima MD, PhD

Authors
  1. Yasuo Koga MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yoshihiko Kitajima MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Atsushi Miyoshi MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ken Sato MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenji Kitahara MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hidenobu Soejima MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kohji Miyazaki MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kohji Miyazaki MD, PhD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Koga, Y., Kitajima, Y., Miyoshi, A. et al. Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers. Ann Surg Oncol 12, 354–363 (2005). https://doi.org/10.1245/ASO.2005.07.020

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1245/ASO.2005.07.020

Keywords

Search

Navigation