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Aberrant DNA methylation has been shown to play an important role during multistage carcinogenesis in various human organs. The aim of the present study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during pancreatic carcinogenesis. Immunohistochemical analysis of DNMT1 in 48 peripheral pancreatic duct epithelia showing no remarkable histological findings without an inflammatory background (DE), 54 peripheral pancreatic duct epithelia with an inflammatory background (DEI), 188 pancreatic intraepithelial neoplasias (PanIN), and 220 areas of invasive ductal carcinoma from surgical specimens resected from 100 patients, was carried out. The average incidence of DNMT1 immunoreactivity increased progressively from DE to DEI (P = 0.003), from DE and DEI to PanIN (P < 0.0001), among PanIN with different grades of dysplasia (from PanIN I to PanIN II, P = 0.0012), from PanIN to invasive ductal carcinomas (P < 0.0001) and among invasive ductal carcinomas with different grades of histological differentiation (from well or moderately to poorly differentiated adenocarcinomas, P < 0.0001). High-level DNMT1 protein expression in invasive ductal carcinomas was correlated significantly with an advanced t category (P = 0.0224) and an advanced stage (P = 0.0294). Moreover, patients with invasive ductal carcinomas showing high-level DNMT1 protein expression had a poorer outcome (P = 0.0469). These data suggest that increased DNMT1 protein expression participates in multistage pancreatic carcinogenesis from the precancerous stage to malignant progression of ductal carcinomas and may be a biological predictor of poor prognosis. (Cancer Sci 2005; 96: 403–408)
Pancreatic cancer is a devastating disease with a very poor prognosis, with a 5-year survival rate of < 3%, and is the fourth or fifth largest cause of cancer-related death worldwide.(1,2) Because ductal carcinomas frequently emerge in pancreases damaged by chronic pancreatitis,(3) at least a proportion of peripheral pancreatic duct epithelia with an inflammatory background may be at the precancerous stage, even though they may show no remarkable histological findings. Recently, Hruban et al.(4) suggested a new nomenclature and classification system for pancreatic intraepithelial neoplasia (PanIN) as a precancerous lesion, and proposed a model of progression from PanIN to ductal carcinoma.(5) Elucidation of genetic and epigenetic alterations in such precancerous conditions and ductal carcinomas showing various clinicopathological features would contribute to a better understanding of the molecular basis of multistage pancreatic carcinogenesis.
DNA methylation plays an important role in transcriptional regulation, chromatin remodeling and genomic stability.(6) Overall DNA hypomethylation and regional DNA hypermethylation are commonly observed in various tumors, including pancreatic cancers.(7,8) Furthermore, accumulating evidence suggests that aberrant DNA methylation is involved even in the early and precancerous stages of human carcinogenesis.(9–15)
DNA methyltransferase 1 (DNMT1) is the major human DNMT(16) and increased levels of its mRNA and protein expression have been reported in several human precancerous conditions and cancers.(17–22) We have reported previously that DNMT1 protein overexpression precedes an increase in the proliferating cell nuclear antigen labeling index in precancerous conditions of the urinary bladder,(21) and is significantly correlated with poorer differentiation of liver(19) and stomach(22) cancers and a poor prognosis in patients with liver cancer.(19) However, to our knowledge, there are no reported data on the expression of DNMT1 at both the mRNA and protein levels in pancreatic cancers. In this study we carried out an immunohistochemical analysis of DNMT1 expression in a large series of precancerous conditions and ductal carcinomas of the pancreas to evaluate its significance in multistage pancreatic carcinogenesis.
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We believe that this is the first report to describe the expression of DNMT1 protein in precancerous conditions and ductal cancers of the pancreas. Initially, the incidence of DNMT1 immunoreactivity increased in DEI compared to DE. Inflammatory cell infiltration around the peripheral pancreatic duct was frequently associated with pancreatic acinar atrophy, suggesting that persistent inflammation had long been present in such areas. Although ductal carcinomas are not always preceded by chronic pancreatitis, and the persistent inflammation observed in a part of our cohort may have been induced secondarily by obstruction of the pancreatic ducts as a result of tumor growth, it is well known that chronic pancreatitis significantly increases the risk of developing pancreatic cancer.(3) Therefore, at least a proportion of DEI may be at the precancerous stage even though they may show no remarkable histological findings. Our data suggest that increased expression of DNMT1 protein may participate even in the very early stage of multistage pancreatic carcinogenesis. It has been reported that some pancreatic ducts showing no remarkable histological findings have K-ras mutation(25) and loss of heterozygosity on some loci.(26) In combination with such genetic aberrations, increased DNMT1 protein expression could play a role in triggering the process of tumorigenesis in ductal epithelia with an inflammatory background. Further studies are needed to understand the molecular mechanisms by which an inflammatory microenvironment affects the level of DNMT1 expression in ductal epithelia.
The incidence of DNMT1 immunoreactivity increased progressively in PanIN compared to DE and DEI and among PanIN that were accompanied by increasing grades of dysplasia, and was further increased in invasive ductal carcinomas, clearly indicating a continuous association of DNMT1 protein overexpression with the progression of multistage pancreatic carcinogenesis. In stomach and colorectal cancers, we have reported that DNMT1 overexpression results in a CpG island methylator phenotype,(20,22) which is defined as frequent DNA hypermethylation on CpG islands that are not methylated in normal tissues.(27) DNA hypermethylation on CpG islands of several cancer-related genes has been reported in PanIN and ductal cancers of the pancreas.(28) By analogy with the findings for stomach and colorectal cancers, DNMT1 protein overexpression may actually result in DNA hypermethylation on such CpG islands of cancer-related genes during pancreatic carcinogenesis. As DNMT1 shows a preference for hemimethylated rather than unmethylated substrates in vitro,(29) and targets replication foci by binding to proliferating cell nuclear antigen,(30) it has been considered to be a maintenance form of DNMT that copies methylation patterns after DNA replication. However, in human cancers, unknown factors may potently target DNMT1 to unmethylated substrate DNA, such as CpG islands of specific genes. Moreover, some workers have proposed that DNMT1 possesses both maintenance and de novo DNA methylation activity in vivo, regardless of its in vitro substrate preference.(31) Therefore, it is feasible that DNMT1 protein overexpression actually results in de novo and regional DNA hypermethylation during carcinogenesis.
In the present study, we noted that the incidence of DNMT1 immunoreactivity increased abruptly from WD to MD and was further increased in PD (Fig. 3). Even in the same patient, PD components showed a much higher incidence of DNMT1 immunoreactivity than WD or MD components (Fig. 2). Accordingly, we detected a significant correlation between high-level DNMT1 protein expression and an advanced t category and advanced stage at diagnosis defined by the Japan Pancreatic Society.(23) However, high-level DNMT1 protein expression did not show a significant correlation with the clinicopathological parameters defined by the UICC classification.(32) This discrepancy may be attributable to the fact that the classification we used(23) reflects in detail the extent of the tumor by using more parameters than the UICC classification.(32) Our data suggest that DNMT1 protein overexpression was associated with aggressiveness of pancreatic cancers. We then analyzed the overall survival curve by the Kaplan–Meier method (Fig. 4). Almost half of the patients died within the first year after surgery, irrespective of their DNMT1 protein expression level. This appears to reflect the devastating nature of pancreatic cancer. However, a significant difference was observed between the patient groups with a high- and a low-level of DNMT1 protein expression: those with high-level DNMT1 protein expression had a worse prognosis from approximately the second year after surgery. Immunohistochemical examination of DNMT1 in cytological, biopsy or surgically resected specimens may be clinically useful for predicting the outcome of patients with pancreatic cancer.
Although DNMT1 is the major DNMT in humans, the activity of DNMT3a and DNMT3b has also been confirmed.(33) DNA methylation patterns are considered to be established through the cooperation of the three DNMT. Further immunohistochemical studies on the cooperative action of DNMT1 with other DNMT in tissue specimens may provide further understanding of the background factors determining the DNA methylation pattern in pancreatic cancers. DNMT may become a molecular target for preventive and therapeutic strategies against multistage pancreatic carcinogenesis.