Tumor progression through epigenetic gene silencing of O(6)-methylguanine-DNA methyltransferase in human biliary tract cancers.
BACKGROUND
We previously demonstrated in an immunohistochemical study that reduced expression of O(6)-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, beta-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.
Koga
Yasuo
Y
Department of Surgery, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501, Japan.
Kitajima
Yoshihiko
Y
Miyoshi
Atsushi
A
Sato
Ken
K
Kitahara
Kenji
K
Soejima
Hidenobu
H
Miyazaki
Kohji
K
D000368:Aged; D000369:Aged, 80 and over; D001650:Bile Duct Neoplasms / Q000235:genetics* / Q000473:pathology; D017734:Bile Ducts, Extrahepatic; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D018450:Disease Progression; D044127:Epigenesis, Genetic / Q000235:genetics*; D005260:Female; D005706:Gallbladder Neoplasms / Q000235:genetics*; D020868:Gene Silencing / Q000502:physiology*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D051176:beta Catenin
C495270:CTNNB1 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D051176:beta Catenin; D019853:O(6)-Methylguanine-DNA Methyltransferase