Carcinogenic risk in the biliary epithelium of children with congenital biliary dilatation via the DNA damage repair pathway

Congenital biliary dilatation (CBD) is a high-risk factor for biliary tract cancer (BTC). We previously reported the potential for carcinogenesis in the biliary epithelium of patients with CBD. In this study, we investigated potential carcinogenetic pathways, focusing on the DNA damage repair response, in children with CBD and compared the findings with those in adults. We enrolled 6 children with CBD and 10 adults with CBD without BTC who underwent extrahepatic bile duct resections, plus 4 control patients who underwent pancreaticoduodenectomy for non-biliary cancer. Levels of phosphorylated histone H2AX (γH2AX), MRE11, and Ku-70 in the biliary tract epithelium were evaluated by immunohistochemistry. The levels of γH2AX, MRE11, and Ku-70 were significantly higher in the gallbladder epithelium and bile duct epithelium of both children and adults than in controls. Children and adults with CBD might develop BTC via the DNA damage repair pathway, as evidenced by increased γH2AX, MRE11, and Ku-70 expression.


Introduction
It is well-known that most cases of congenital biliary dilatation (CBD) are accompanied by pancreaticobiliary maljunction (PBM).PBM is a congenital abnormality in which the bile and pancreatic ducts merge outside the wall of the duodenum and usually have a long common channel.This anatomical anomaly reportedly causes reflux of pancreatic juice and bile in the pancreaticobiliary duct, which then produces toxic substances (e.g.activated pancreatic enzymes), induces repeated damage and repair of the biliary tract mucosa, exacerbates histological changes, and eventually causes biliary tract carcinogenesis through various gene mutations [1,2].This sequence of changes is referred to as the "hyperplastic-dysplastic-cancer sequence".Thus, CBD has a high risk of causing biliary tract cancer (BTC) [3].
Many researchers have been trying to elucidate the carcinogenic mechanism from various perspectives.Toxic substances damage the biliary epithelium, multiple changes in oncogenes and tumor suppressor genes occur during the repair process, and cancer is caused by the interactions among these various altered genes [4][5][6].We previously used metabolomics to examine changes in the metabolites present in bile juice due to the reflux of pancreatic juice into the biliary tract, which is considered to be the cause of carcinogenesis in PBM.That study identified lysophosphatidylcholine and triacylglycerol as metabolites that contributed to carcinogenesis among those commonly increased in PBM and BTC compared with controls [7].
Lysophosphatidylcholine itself reportedly induces the production of reactive oxygen species (ROS) in cholangiocytes and induces cancer-related genes through senescence, which is associated with biliary carcinogenesis [8].It has also been reported that ROS are produced in epithelial cells under inflammatory conditions through the induction of triacylglycerol, which further induces mitochondrial dysfunction [9], suggesting that ROS might be involved in PBM carcinogenesis.In addition, elevated levels of DNA damage have been reported in chronic inflammation compared with acute inflammation, and genetic changes occur during the process of repairing this damage, leading to carcinogenesis [10].ROS are also reportedly induced in the gallbladder epithelium of patients with PBM under chronic inflammation [11].Furthermore, our previous results showed that KRAS expression was enhanced in both the gallbladder and bile duct in PBM compared with controls, suggesting the possibility of accumulated oncogenic changes [12].
Enhanced ROS production with insufficient scavenging capacity results in cellular damage, including DNA singleand double-strand breaks (DSBs) [10].The DNA repair pathway is then induced to repair DSBs [13].In brief, the histone H2AX of the H2A subtype (γH2AX) is phosphorylated on Ser-139, which occurs in the early stages of the DSB response [14,15].γH2AX is regarded as a general marker of DSBs.In addition, γH2AX activates DNA damage repair pathways by interacting with DSB repair proteins, such as MRE11 and KU70, which are also markers of the DSB repair response [16,17].
Therefore, we hypothesized that the metabolites we previously identified might cause ROS-induced oxidative DNA damage and activate the DNA repair pathways, causing the accumulation of cancer-related genetic changes during the repair process, which leads to carcinogenesis in biliary epithelial cells of both adults and children with CBD under chronic inflammation.
In the present study, we investigated the carcinogenic mechanism of BTC, focusing on the involvement of the DNA damage repair pathway, in the biliary tract epithelium of children with CBD and compared these findings with those in adult patients without BTC.

Patients
In this study, we enrolled 6 children with CBD (children group; C group, n = 6) and 10 adults with CBD but not BTC (adult group; A group, n = 10) who underwent total resection of their extrahepatic biliary tract with biliary reconstruction.We also included a control group (Cont.group, n = 4) of patients who underwent pancreaticoduodenectomies and did not have BTC.All patients had no history of cholangitis.The detailed characteristics of the patients are shown in Table 1.
The protocol for this study was approved by the Ethics Committee of Tokushima University Hospital (No. 3010) and conformed with the provisions of the Declaration of Helsinki.All patients gave their informed consent for surgery and study participation.
The central part of bile duct specimens was selected to evaluate the expression of γH2AX, MRE11, and KU70.Each section was viewed in 5 randomly selected 200 × magnification fields to determine the expression patterns.Expression of these proteins was evaluated as the percentage of each epithelium sample that showed positive staining per field.Expression of γH2AX, MRE11 and KU70 in the gallbladder and bile duct epithelium was compared among groups for each epithelial type.

Statistical analyses
Data for γH2AX, MRE11, and KU70 are presented as the mean ± standard deviation.Comparisons of several parameters between two groups were made using Bonferroni's multiple comparison test.All statistical analyses were performed using the JMP 8.0.1.statistical software program (SAS Institute, Cary, NC, USA).P < 0.05 was considered significant.

Discussion
It is well-known that CBD patients with chronic inflammation have a high incidence of biliary tract cancer [1,18,19].Genetic alterations in the biliary epithelium of CBD patients have been investigated [20][21][22], but the mechanism of carcinogenesis is still unknown.In addition, few similar studies have been performed on the biliary epithelium of pediatric CBD patients.
Our previous metabolomics analysis identified carcinogenesis-related metabolites in the bile of PBM patients [7].In that study, we investigated the possibility that these metabolites might cause DNA damage via ROS production in biliary epithelial cells, inducing activation of DNA damage repair pathways.We also previously reported that biliary epithelial cells in pediatric CBD had carcinogenic potential [12] and investigated whether or not similar carcinogenic events might occur in children and adults.The results showed that DNA damage markers and DNA damage repair response markers were significantly elevated in adult and pediatric CBD patients compared with controls, suggesting the involvement of DNA damage repair pathways in the carcinogenic mechanisms of CBD.Furthermore, pediatric CBD patients also showed a significant increase in these DNA damage and DNA repair markers compared with controls, and the increase was comparable to that in adult CBD patients, suggesting that CBD has the same carcinogenic potential in children as in adults.
Chronic inflammation is generally thought to promote carcinogenesis due to oxidative stress [23].Frick et al. also reported that DSBs were a core molecular lesion of inflammation-induced carcinogenesis and that oxidative DNA damage due to inflammation was the driving force of mutagenesis [24].Under the chronic inflammatory conditions of inflammatory bowel disease, oxidative DNA damage is increased with the degree of inflammation; the expression of DSB repair proteins is likewise increased with the grade of inflammation and dysplasia, and elevated DNA damage with DNA damage repair is associated with the accumulation and spread of precancerous lesions [24].Therefore, under chronic inflammatory conditions, there is increased oxidative stress on the intestinal epithelium, and this oxidative stress increases not only with the degree of inflammation but also as the disease progresses toward carcinogenesis.
DNA damage and repair responses are also enhanced during the carcinogenesis process, which is similar to our results and supports our findings.Furthermore, activation of the DNA damage repair pathway may promote the cell survival and the accumulation of genetic alterations that lead to inflammation-induced carcinogenesis.Biliary epithelial cells in CBD under chronic inflammatory stress have a high proliferation rate and are constantly exposed to replicative stress, which may also contribute to increased DSBs [10,25].
At the beginning of the present study, it was assumed that the expression of DNA damage repair response genes would gradually increase in proportion to the duration of exposure to inflammation; however, DNA damage repair response genes were unexpectedly highly expressed in children as well as adults, regardless of the duration of exposure to pancreatic juice reflux.This finding suggests that disruption of the DNA damage repair response may be an early response of the biliary carcinogenesis mechanism and may lead to subsequent genetic changes associated with carcinogenesis.Through their in vitro studies, Chang et al. reported that the addition of cytotoxic substances induced intracellular ROS production that led to DNA strand breaks, which in turn induced aggressive carcinogenesis through oncogenic RAS [26].It is possible that the expression and activation of KRAS that we previously observed in CBD patients [12] was one such accumulated change in cancer-related genes from the oxidative DNA damage repair response that we examined in this study of CBD patients.Furthermore, that genetic change in K-ras was observed in pediatric cases [12], which may support the involvement of the high expression of DNA damage repair response genes in the early stages of the biliary tract carcinogenesis mechanism.
In general, the DSB repair pathways include homologous recombination (HR) or non-homologous end joining (NHEJ).Compared with HR, NHEJ is considered to be more error-prone, as it directly ligates truncated DNA ends without using a homologous template.NHEJ is active in all cell cycle states and is thought to be more effective than HR but at the cost of creating more DNA mutations [27].Our findings suggested that NHEJ is activated in CBD biliary epithelial cells, as KU70, which binds to the ends of DSBs and is required for NHEJ, is elevated, suggesting that this pathway may be a source of mutations in CBD.
In summary, our results suggest that, in the chronic inflammatory condition of CBD, lysophosphatidylcholine and triacylglycerol in the bile induce ROS generation in the biliary epithelium, which leads to oxidative DNA damage and activation of DNA repair pathways.This process of chronic DNA damage and repair eventually induces the expression and activation of KRAS, which can lead to carcinogenesis.
However, several limitations associated with the present study warrant mention.First, the number of cases was small.Second, the number of controls was small as well, and all were adults.We did not have the opportunity to obtain normal gallbladder and bile duct specimens from children and could not use pediatric cases as controls.Finally, it will be necessary to examine patients with both BTC and CBD in the future.
In conclusion, the expression of γH2AX, MRE11, and KU70 in the biliary epithelium, which are markers of the activation of the DNA damage repair pathway, may influence carcinogenesis in CBD and predispose both children and adults with CBD to develop BTC.These findings suggest that both children and adults with CBD may develop BTC through overactivation of the DNA damage repair pathway in response to cellular ROS.

Table 1
Cys cystic dilatation of the common bile duct, Spi spindle dilatation of the common bile duct, AMY amylase, CBD congenital biliary dilatation, C children with CBD, A adults with CBD * Age at the operation, **A history of cholangitis