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

doi:10.21203/rs.3.rs-1426866/v1

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

https://doi.org/10.21203/rs.3.rs-1426866/v1

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Background: Congenital biliary dilatation (CBD) is a high-risk factor for biliary tract cancer (BTC). We have 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 adults.

Methods: We enrolled six children with CBD and 10 adults with CBD without BTC who underwent extrahepatic bile duct resections, plus four 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.

Results: Levels of γH2AX, MRE11, and Ku-70 were significantly higher in the gallbladder epithelium and bile duct epithelium of both children and adults compared with in controls.

Conclusions: Children and adults with CBD might develop BTC via the DNA damage repair pathway, as evidenced by increased γH2AX, MRE11, and Ku-70 expression.

congenital biliary dilatation

carcinogenesis

DNA damage repair pathway

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. It is reported that this anatomical anomaly causes reflux of pancreatic juice and bile in the pancreaticobiliary duct, produces toxic substances such as activated pancreatic enzymes and so on, repeats damage and repair to the biliary tract mucosa, progresses histological changes, and eventually biliary tract carcinogenesis occurs through various gene mutations [1, 2]. This sequence of changes is referred as the “hyperplastic-dysplastic-cancer sequence”. Thus, CBD has a high-risk potential for the incidence of 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 [4–6]. 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. This study identified lysophosphatidylcholine and triacylglycerol as metabolites that contribute to carcinogenesis among the metabolites commonly increased in PBM and BTC compared with controls [7]. It has been reported that lysophosphatidylcholine itself 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 is produced in epithelial cells under inflammatory conditions through the induction triacylglycerol, which further induces mitochondrial dysfunction [9], suggesting that ROS might be involved in PBM carcinogenesis. Additionally, higher levels of DNA damage have been reported in chronic inflammation compared with in acute inflammation, and genetic changes occur during the process of repairing this damage, leading to carcinogenesis [10]. Furthermore, it has been reported that ROS is actually induced in the gallbladder epithelium of patients with PBM under chronic inflammation [11]. Moreover, 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].

Finally, enhanced ROS production with insufficient scavenging capacity results in cellular damage, including DNA single- and double-strand breaks (DSBs) [10]. The DNA repair pathway is then induced to repair DSBs [13]. Briefly, 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. Additionally, 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 an 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 this study, we investigated the carcinogenic mechanism of BTC, focusing on 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 six 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 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.

Immunohistochemistry

Excised gallbladder and bile duct specimens were fixed in 10% formaldehyde neutral buffer solution and embedded in paraffin. Immunohistochemical staining was performed on 4 µm-thick sections using a rabbit monoclonal anti-γH2AX antibody (Cell Signaling Technology, Inc., Danvers, MA, USA, #9718; diluted 1:240 in PBS), a rabbit polyclonal anti-Mre11 antibody (Cell Signaling Technology, Inc., #4895; diluted 1:500 in PBS), and a mouse monoclonal anti-Ku70 antibody (Santa Cruz Biotechnology, Inc, Dallas, TX, USA, sc-17789; diluted 1:50 in PBS). After incubating the sections with primary antibody at 4°C overnight, antigen visualization was performed by indirect immunoperoxidase staining using avidin–biotin complexes (Dako, Glostrup, Denmark) and DAB Tablets (Wako Pure Chemical Industries, Ltd., Osaka, Japan). In gallbladder and bile duct epithelium, brown nuclei were considered to indicate positive staining (γH2AX⁺, MRE11⁺, and KU70⁺). The central part of bile duct specimens was selected to evaluate the expression levels of γH2AX, MRE11, and KU70. Each section was viewed in five 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 levels for γH2AX, MRE11 and KU70 in the gallbladder and bile duct epithelium were compared among each group in each epithelial type.

Statistical analysis

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

γH2AX, MRE11 and KU70 expression in gallbladder epithelium

In the gallbladder epithelium, γH2AX expression was significantly higher in both children (56.7% ± 13.7%) and adults (62.0% ± 12.5%) with CBD compared with in controls (3.8% ± 4.8%); γH2AX levels were similar between children and adults with CBD (Fig. 1). MRE11 expression was significantly elevated in both children (63.3% ± 17.8%) and adults (69.5% ± 11.9%) with CBD compared with controls (16.3% ± 11.1%); MRE11 levels were similar between children and adults with CBD (Fig. 2). KU70 expression was significantly higher in both children (60.8% ± 18.3%) and adults (65.0% ± 18.3%) with CBD compared with controls (15.5% ± 10.5%); KU70 levels were similar between children and adults with CBD (Fig. 3).

γH2AX, MRE11 and KU70 expression in bile duct epithelium

In the bile duct epithelium, γH2AX expression was significantly higher in both children (62.5% ± 9.9%) and adults (67.0% ± 8.2%) with CBD compared with controls (3.3% ± 4.7%); γH2AX expression was similar between children and adults with CBD (Fig. 4). Compared with controls (13.8% ± 13.8%), MRE11 expression was significantly elevated in both children (64.2% ± 14.3%) and adults (72.0% ± 12.8%) with CBD; MRE11 expression was similar between children and adults with CBD (Fig. 5). Furthermore, KU70 expression was significantly elevated in both children (66.7% ± 19.7%) and adults (62.0% ± 13.8%) with CBD compared with controls (12.5% ± 10.4%); KU70 expression was similar between children and adults with CBD (Fig. 6).

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 [20–22], but the mechanism of carcinogenesis is still unknown. Additionally, 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 this study, we investigated the possibility that these metabolites may cause DNA damage via ROS production in biliary epithelial cells, inducing activation of DNA damage repair pathways. Additionally, we have previously reported that biliary epithelial cells in pediatric CBD have carcinogenic potential [12] and have also investigated whether similar carcinogenic mechanisms 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 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]. In other words, in chronic inflammatory conditions, there is increased oxidative stress on the intestinal epithelium, and this oxidative stress is enhanced 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 cell survival and accumulate 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].

From a set of 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.

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 because 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 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 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.

In conclusion, the expression of γH2AX, MRE11, and KU70 in the biliary epithelium, which are also markers for activation of the DNA damage repair pathway, could influence carcinogenesis in CBD and predispose both children and adults with CBD to develop BTC. These findings suggested that both children and adults with CBD may develop BTC through overactivation of the DNA damage repair pathway in response to cellular ROS.

BTC: biliary tract cancer; CBD: congenital biliary dilatation; DSB: DNA double-strand break; γH2AX: phosphorylated histone H2AX; HR: homologous recombination; NHEJ: non-homologous end joining; PBM: pancreaticobiliary maljunction; ROS: reactive oxygen species; SD: standard deviation

Acknowledgments

We thank James P. Mahaffey, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Conflict of interest: The authors declare that they have no conflicts of interest.

Funding: None

Tsuchida A, Itoi T, Aoki T, Koyanagi Y. Carcinogenetic process in gallbladder mucosa with pancreaticobiliary maljunction. Oncol Rep. 2003; 10(6): 1693–1699.
Funabiki T, Matsubara T, Miyakawa S, Ishihara S. Pancreaticobiliary maljunction and carcinogenesis to biliary and pancreatic malignancy. Langenbecks Arch Surg. 2009; 394(1): 159–169.
Tashiro S, Imaizumi T, Ohkawa H, Okada A, Katoh T, Kawaharada Y, et al. Pancreaticobiliary maljunction: retrospective and nationwide survey in Japan. J Hepatobiliary Pancreat Surg. 2003; 10(5): 345–351.
Matsubara T, Sakurai Y, Zhi L-Z, Miura H, Ochiai M, Funabiki T. K-ras and p-53 gene mutations in noncancerous biliary lesions of patients with pancreatico biliary maljunction. J Hepatobiliary Pancreat Surg. 2002; 9(3): 312–321.
Nagai M, Watanabe M, Iwase T, Yamao K, Isaji S. Clinical and genetic analysis of noncancerous and cancerous biliary epithelium in patients with pancreaticobiliary maljunction. World J Surg. 2002; 26(1): 91–98.
Kamisawa T, Funata N, Hayashi Y, Egawa N, Nakajima H, Tsuruta K, et al. Pathologic changes in the non-carcinomatous epithelium of the gallbladder in patients with a relatively long common channel. Gastrointest Endosc. 2004; 60(1): 56–60.
Mori H, Morine Y, Mawatari K, Chiba A, Yamada S, Saito YU, Ishibashi H, Takahashi A, Shimada M. Bile Metabolites and Risk of Carcinogenesis in Patients With Pancreaticobiliary Maljunction: A Pilot Study. Anticancer Res. 2021; 41(1): 327–334.
Shimizu R, Kanno K, Sugiyama A, Ohata H, Araki A, Kishikawa N, Kimura Y, Yamamoto H, Kodama M, Kihira K, Tazuma S. Cholangiocyte senescence caused by lysophosphatidylcholine as a potential implication in carcinogenesis. J Hepatobiliary Pancreat Sci. 2015 Sep; 22(9): 675–682.
Fock E, Bachteeva V, Lavrova E, Parnova R. Mitochondrial-Targeted Antioxidant MitoQ Prevents E. coli Lipopolysaccharide-Induced Accumulation of Triacylglycerol and Lipid Droplets Biogenesis in Epithelial Cells. J Lipids. 2018 Sep 2; 2018: 5745790.
Kiraly O, Gong G, Olipitz W, Muthupalani S, Engelward BP. Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo. PLoS Genet. 2015 Feb 3; 11(2): e1004901.
Otani K, Shimizu S, Chijiiwa K, Yamaguchi K, Noshiro H, Tanaka M. Immunohistochemical detection of 8-hydroxy-2'-deoxyguanosine in gallbladder epithelium of patients with pancreaticobiliary maljunction. Eur J Gastroenterol Hepatol. 2001 Nov; 13(11): 1363–1369.
Mori H, Masahata K, Umeda S, Morine Y, Ishibashi H, Usui N, Shimada M. Risk of carcinogenesis in the biliary epithelium of children with congenital biliary dilatation through epigenetic and genetic regulation. Surg Today. 2021 Jun 16. doi: 10.1007/s00595-021-02325-2. Online ahead of print.
Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009 Oct 22; 461(7267): 1071–1078.
Firsanov DV, Solovjeva LV, Svetlova MP. H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues. Clin Epigenet. 2011 Aug; 2(2): 283–297.
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998 Mar 6; 273(10): 5858–5868.
Chapman JR, Taylor MR, Boulton SJ. Playing the end game: DNA double-strand break repair pathway choice. Mol Cell. 2012 Aug 24; 47(4): 497–510.
Kuhfittig-Kulle S, Feldmann E, Odersky A, Kuliczkowska A, Goedecke W, Eggert A, et al. The mutagenic potential of non-homologous end joining in the absence of the NHEJ core factors Ku70/80, DNA-PKcs and XRCC4-LigIV. Mutagenesis. 2007 May; 22(3): 217–233.
Matsuda T, Marugame T, Kamo K, Katanoda K, Ajiki W, Sobue T; The Japan Cancer Surveillance Research Group. Cancer incidence and incidence rates in Japan in 2003: based on data from 13 population-based cancer registries in the Monitoring of Cancer Incidence in Japan (MCIJ) project. Japanese Journal of Clinical Oncology. 2009; 39(12): 850–858.
Morine Y, Shimada M, Takamatsu H, Araida T, Endo I, Kubota M, et al. Clinical features of pancreaticobiliary maljunction: update analysis of 2nd Japan-nationwide survey. J Hepatobiliary Pancreat Sci. 2013; 20(5): 472–480.
Tsuchida A, Itoi T. Carcinogenesis and chemoprevention of biliary tract cancer in pancreaticobiliary maljunction. World J Gastrointest Oncol. 2010; 2(3): 130–135.
Singh MK, Chetri K, Pandey UB, Kapoor VK, Mittal B, Choudhuri G. Mutational spectrum of K-ras oncogene among Indian patients with gallbladder cancer. J Gastroenterol Hepatol. 2004; 19(8): 916–921.
Tazuma S, Kajiyama G. Carcinogenesis of malignant lesions of the gall bladder: The impact of chronic inflammation and gallstones. Langenbecks Arch Surg. 2001; 386(3): 224–229.
Irrazabal T, Thakur BK, Kang M, Malaise Y, Streutker C, Wong EOY, Copeland J, Gryfe R, Guttman DS, Navarre WW, Martin A. Limiting oxidative DNA damage reduces microbe-induced colitis-associated colorectal cancer. Nat Commun. 2020 Apr 14; 11(1): 1802.
Frick A, Khare V, Paul G, Lang M, Ferk F, Knasmüller S, Beer A, Oberhuber G, Gasche C. Overt Increase of Oxidative Stress and DNA Damage in Murine and Human Colitis and Colitis-Associated Neoplasia. Mol Cancer Res. 2018 Apr; 16(4): 634–642.
Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature. 2005 Apr 14; 434(7035): 907–913.
Chang YJ, Tseng CY, Lin PY, Chuang YC, Chao MW. Acute exposure to DEHP metabolite, MEHP cause genotoxicity, mutagenesis and carcinogenicity in mammalian Chinese hamster ovary cells. Carcinogenesis. 2017 Mar 1; 38(3): 336–345.
Guirouilh-Barbat J, Huck S, Bertrand P, Pirzio L, Desmaze C, Sabatier L, et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol Cell. 2004 Jun 4; 14(5): 611–623.

No competing interests reported.

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

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Version 1

Abstract

Figures

Introduction

Methods

Patients

Immunohistochemistry

Statistical analysis

Results

γH2AX, MRE11 and KU70 expression in gallbladder epithelium

γH2AX, MRE11 and KU70 expression in bile duct epithelium

Discussion

Abbreviations

Declarations

References

Additional Declarations

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