Best Practice & Research Clinical Gastroenterology 29 (2015) 233e244

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Best Practice & Research ClinicalGastroenterology

2

Pathogenesis of cholangiocarcinoma: Fromgenetics to signalling pathways

Sarinya Kongpetch, PhD, Research Fellow, Lecturer a, b, c,Apinya Jusakul, PhD, Research Fellow a, c,Choon Kiat Ong, PhD, Senior Scientist a, c,Weng Khong Lim, PhD, Research Fellow a, c,Steven G. Rozen, PhD, Associate Professor c, d,Patrick Tan, MD, PhD, Professor c, e, f,Bin Tean Teh, MD, PhD, Professor a, c, f, *

a Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore,Singaporeb Department of Pharmacology, Faculty of Medicine and Liver Fluke and Cholangiocarcinoma ResearchCenter, Khon Kaen University, Khon Kaen, Thailandc Division of Cancer and Stem Cell Biology, Duke-National University of Singapore (NUS) Graduate MedicalSchool, Singapored Centre for Computational Biology, Duke-NUS Graduate Medical School, Singaporee Genome Institute of Singapore, Singaporef Cancer Science Institute of Singapore, National University of Singapore, Singapore

Keywords:CholangiocarcinomaMolecular pathogenesisGenetic alterationChromatin

* Corresponding author. Laboratory of Cancer EpSingapore. Tel.: þ65 66 011324.

E-mail addresses: sarinya.kongpetch@nccs.com(A. Jusakul), cmrock@nccs.com.sg (C.K. Ong), w(S.G. Rozen), gmstanp@duke-nus.edu.sg (P. Tan),

http://dx.doi.org/10.1016/j.bpg.2015.02.0021521-6918/© 2015 Elsevier Ltd. All rights reserved

a b s t r a c t

Cholangiocarcinoma (CCA) is a malignant tumour of bile ductepithelial cells with dismal prognosis and rising incidence. Chronicinflammation resulting from liver fluke infection, hepatitis andother inflammatory bowel diseases is a major contributing factorto cholangiocarcinogenesis, likely through accumulation of serialgenetic and epigenetic alterations resulting in aberration of on-cogenes and tumour suppressors. Recent studies making use ofadvances in high-throughput genomics have revealed the geneticlandscape of CCA, greatly increasing our understanding of its un-derlying biology. A series of highly recurrent mutations in genes

igenome, Division of Medical Sciences, National Cancer Centre Singapore,

.sg, sarinyako@kku.ac.th (S. Kongpetch), apinya.jusakul@duke-nus.edu.sgengkhong.lim@duke-nus.edu.sg (W.K. Lim), steve.rozen@duke-nus.edu.sgteh.bin.tean@singhealth.com.sg (B.T. Teh).

.

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such as TP53, KRAS, SMAD4, BRAF, MLL3, ARID1A, PBRM1 and BAP1,which are known to be involved in cell cycle control, cell signallingpathways and chromatin dynamics, have led to investigations oftheir roles, through molecular to mouse modelling studies, incholangiocarcinogenesis. This review focuses on the landscapegenetic alterations in CCA and its functional relevance to the for-mation and progression of CCA.

© 2015 Elsevier Ltd. All rights reserved.

Introduction

Cholangiocarcinoma (CCA) is a lethal malignancy with poor prognosis that makes up 10e25% of allprimary liver cancers diagnosedworldwide. Its incidence is highest in northeasternThailand, borderingLaos and Cambodia, with very high age-standardized incidence rates (ASRs) of 84.6 and 36.8 per100,000 in males and females, respectively [1]. This is in contrast to ASRs of less than 1.5 per 100,000 inWestern countries [2]. Several risk factors for CCA are related to geography and etiology. For instance,infestation of liver flukes such as Opisthorchis viverrini (Ov) and Clonorchis sinensis has been associatedwith the carcinogenesis of CCA, especially in countries lining the Mekong River such as Thailand,Vietnam, and Laos [3]. Hepatolithiasis is also a common risk factor for CCA, particularly intrahepatic CCA(ICC) in Asian countries. Moreover, patients with hepatolithiasis are also likely to have liver flukeinfestation [4]. Cirrhosis, hepatitis B (HBV) and hepatitis C viral (HCV) infection among other risk factorsidentified frommeta-analysis [5]. In contrast, primary sclerosing cholangitis (PSC) is the most commonrisk factor of CCA in the Western countries. The well-established association between PSC and CCA ismarked by chronic inflammation, resulting in liver injury and likely proliferation of the progenitor cells[6]. Other potential contributing factors to ICC include HIV infection, inflammatory bowel disease in-dependent of PSC, alcohol, smoking, fatty liver disease, cholelithiasis and choledocholithiasis [7e9].Together, all these known risk factors point to a common role for chronic biliary inflammation in CCA.

In this review, we focus on recurrent alterations in the genetic landscape of CCA. The spectrum andfrequency of these alterations, including those identified from recent whole-exome sequencingstudies, indicate the possible involvement of their associated molecular pathways in CCA. This isfurther substantiated by in vitro and in vivo studies. The results from these experiments, especially thelatter, have potential clinical implications as they point to the importance of targeting specific alteredpathways in each CCA in improving patient outcomes.

Tumour biology and cells of origin

CCA is an epithelial malignant tumour arising from different locations of the biliary tree. It can becategorized into two common groups by anatomical location; intrahepatic (ICC) and extrahepaticcholangiocarcinoma (ECC). ICC refers to tumours arising from the large and small bile ducts within theliver. ECC, on the other hand, refers to bile duct tumours arising outside the liver, that can be furtherdivided into perihilar and distal CCAs, separated by the junction of cystic and common bile ducts [8].The traditional classification of ICC includes well, moderately and poorly differentiated adenocarci-nomas. Recently, there is a new pathological concept to classify ICC into conventional ICC, bile ductularICC, intraductal neoplasms and rare variants (combined hepatocellular CCA, undifferentiated type,squamous/adenosquamous type) [10]. Interestingly, a marker of hepatic progenitor cells has beendetected in the bile ductular and combined hepatocellular CCA types, suggesting these may haveoriginated from hepatic progenitor cells [11,12]. Recent studies also propose that rather than being ofsingle cellular origin, CCA may have developed from a combination of cholangiocyte, the peribiliarygland around bile duct, hepatic progenitor cell or hepatocytes [8]. Mouse models have shown thattransformed hepatocytes, hepatoblasts, and hepatic progenitor cells are capable of producing a broadspectrum of liver malignancies ranging from CCA to hepatocellular carcinoma (HCC) [13]. These studies

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suggest that cholangiocytes alone may not be sufficient for CCA carcinogenesis, and that this processmay involve the transformation of multiple cell types. In one study, Sekiya et al reported that hepa-tocytes can transform into biliary cells through the Notch pathway, leading to ICC formation [14]. Inanother, Fan et al showed that overexpression of both NOTCH1 and AKT leads to lethal ICC formation,again via transformation of hepatocytes into cholangiocyte precursors [15], although AKT over-expression alone may not be sufficient for ICC formation [16]. More recently, it was shown that miceengineered to express both mutant IDH2 and KRAS in the adult liver displayed phenotypes such as theexpansion of liver progenitor cells, development of premalignant biliary lesions, and finally progres-sion to metastatic ICC [17].

Molecular and cellular pathogenesis

As alluded to above, chronic infection and inflammation in the bile ducts play an important role incholangiocarcinogenesis. Inflammation causes the release of proinflammatory cytokines leading toinduction of nitric oxide synthase (iNOS), a generator of nitric oxide (NO) in cholangiocytes. NO pro-duced in infected and inflamed tissues has been postulated to contribute to epithelial cell carcino-genesis by causing damage to DNA and proteins [18]. NO can also directly oxidize DNA, resulting inmutagenic changes [19] and stimulates cyclooxygenase-2 (COX-2) expression promoting cholangiocytegrowth via activation of growth factors such as EGFR, MAPK, and IL-6 [20]. In the hamster CCA model,chronic inflammation triggered by repeated Ov infection was reported to mediate iNOS-dependentDNA damage in intrahepatic bile duct epithelium and inflammatory cells, and the combination of Ovinfection and exposure of nitrosamine led to development of CCA [21,22]. Obviously, advances incancer genomics as a result of more effective and high-throughput profiling technologies have allowedcharacterization of the genetic alterations, including their spectrum and frequency, in CCA associatedwith different etiological factors.

Chromosomal changes

Several studies have described chromosomal aberrations in CCA. A meta-analysis of comparativegenomic hybridization studies identified common chromosomal gains at 1q, 5p, 7p, 8q, 17q and 20q aswell as losses at 1p, 4q, 8p, 9p, 17p and 18q [23]. Patterns of genomic changes reflect differences inrelation to ethnicity and etiology. Tumour samples from Asian countries reveal common patterns ofgains in chromosomes 5p, 6p, 7p, 8q, 11q, 13q, 17q, and 20q and losses at 4q, 6q, 8p, 10p, 17q, 18q, and22q [24,25], whereas karyotyping of European CCA cases showed greater diversity. The only regionsshared by European tumours were gains in 7p and 8q, and losses in 1p, 4q, and 9p [26]. Furthermore,recurrent chromosomal gains at 1q, 8q and 17q and losses at 4q, 8p and 17p were reported in both CCAand HCC, implying that there may be a close relationship between these two cancer types [23].

In a separate study, significant gains of 2p, 5p, 22q and significant losses of 8q, 10q, 11p, and 18qwere observed in CCA and these chromosomal regions contained approximately 153 genes, some ofwhich may serve as oncogenes or tumour suppressor genes including those involved in JAK-STAT andMAPK pathways. Other studies also showed gains and losses of chromosomal regions containingcancer-driving genes such as ERBB2/HER2 on 17q, MAP2K2/MEKs on 19p, EGFR on 7p12, PDGFA on7p22, CDKN2A on 9p21 and TP53 on 17p13 [27e29]. Interestingly, copy number gains at 5p15.33 and22q13.33 were correlated with early systemic recurrence and poor disease-free survival in CCA [30].Several studies also described chromosomal aberrations in Ov-related CCA, including gain of 21q22 andlosses of 1p36, 9p21, 17q13 and 22q12 [31e33].

Aberrant epigenetic landscape

Epigenetic dysregulation including histone modification and DNA methylation has been implicatedin the pathogenesis of many cancers including CCA. In tumours, the aberrant DNA methylation occursat the 50 methylcytosine (5-mc) in CpG rich area in the promotor region of tumour suppressor genesleading to their transcriptional silencing. Hypermethylation of p16INK4a/CDKN2A (17e83%), p15INK4b(54%), p14ARF (19e30%), RASSF1A (31e69%), and APC (27e47%) were found in CCA [34e37]. In a study of

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36 CCA cases, TP53mutationwith hypermethylated promoter of p14ARF, DAPK, and/or ASC appeared tocontribute to more aggressive CCA and shorter survival [38].

Epigenetic changes in the genes linked to cytokine and other signalling pathways have also beenimplicated in CCA. For example, the promoter of SOCS3, which is the upstream regulator of JAK/STATcytokine signalling was frequently hypermethylated in CCA [35,39]. The Wnt signalling modulator,SFRP1 was also hypermethylated in CCA at frequencies as high as 85% [40]. On the other hand,hypermethylation of SFRP2 promoter leading to its lower expression, was correlated with poorprognosis [41]. In the future, epigenomic profiling of CCA including histone marks, promoters andenhancers may further shed light on CCA tumorigenesis and progression.

microRNAs (miRNAs) dysregulation

miRNAs are small noncoding RNA that are approximately 20e22 nucleotides in length. Theynegatively regulated target gene expression by binding to 30UTR sites, leading to translational inhi-bition as well as mRNA degradation. Dysregulated miRNAs have been implicated in cancer develop-ment including CCA tumorigenesis. These miRNAs regulating oncogenes (onco-miRNAs) are involvedin biological processes, from cell cycle, apoptosis to cancer metabolism at the post-transcriptional level[42,43]. A comprehensive profiling of miRNA in CCA cell lines (HUCCT1 and MEC) revealed biliaryepithelial cell-specific miRNAs, i.e., miR22, miR125a, miR127, miR199a, miR199a*, miR214, miR376aand miR424, which are downregulated in these lines [44]. In a separate study, miR21 was found to beupregulated in ICC compared to normal epithelial bile duct tissue. Inhibition of miR21 was shown toincrease protein expression of PDCD4 and TIMP3 which are the inhibitors of program-cell death andmetastasis, respectively [45]. Moreover, miR21 was shown to stimulate CCA cell growth and resistanceto chemotherapy by inhibiting PTEN, a tumour suppressor [46]. Other studies have shown that miR25has an anti-apoptotic effect in CCA via inhibiting the death receptor, TRAIL (TNF-related apoptosis-inducing ligand) [47] whereas miR26a, acting on its downstream GSK-3b, could mediate intracellularaccumulation of b-catenin, promoting proliferation and colony formation in cholangiocarcinoma [43].Other dysregulated miRNAs in CCA include miRlet7a (activator of STAT3 signalling pathway) andmiR421 (suppressor of tumour suppressor gene FXR), and these have been shown to regulate cellproliferation, colony formation and migration [48,49].

Structural variation driving cholangiocarcinogenesis

There is emerging evidence of the involvement of novel genomic rearrangements in epithelialcancers such as CCA. These genomic rearrangements include gene amplifications, chromosomaltranslocations, inversions and deletions. They may represent polymorphisms that are neutral infunction, or convey phenotypes such as changing the copy number, disrupting genes and creatingfusion genes [50]. Gene fusions resulting from chromosomal rearrangements are one of the mostcommon events, often considered as ‘onco-fusion proteins’ in cancer development [51,52]. Many fusionkinases with active kinase domains have been associated with tumour initiation via activation ofdownstream kinases leading to progressive phenotypes in cancer (Fig. 1). Tyrosine kinase gene fusionssuch as ROS and FGFR gene fusions with intact kinase domains were identified in various cancer types.ROS1 translocation was reported in 9% of CCA patients [53]. Later on, a mouse model habouring FIG-eROS1 fusion gene that eventually promoted ICC development was generated [54]. More recently, RNAsequencing studies have reported FGFR2 gene fusions in CCA tumours [55e57]. Importantly, suchfusion proteins may serve as potential therapeutic targets for FGFR inhibitors. One study reportedFGFR2-AHCYL1 and FGFR2-BICC1 which are mutually exclusive to KRAS/BRAF/ROS1 alterations [55]while the other found FGFR2-BICC1, FGFR2-MGEA5 and FGFR2-TACC3 fusions [56,57]. Furthermore,overexpression of the FGFR2 fusions and FGFR3 fusion resulted in altered cell morphology and increasedcell proliferation. In vitro and in vivo studies demonstrated increasing sensitivity to FGFR inhibitors inmouse fibroblast and bladder cancer cell lines that haboured FGFR fusions [55,57]. Finally, treatmentwith FGFR inhibitors such as pazopanib and ponatinib in patients has shown improved clinical re-sponses in CCA habouring FGFR2 gene fusions, although the study was limited in terms of cohort size[56]. To date, the full spectrum and frequency of genomic rearrangements in CCA, especially of different

Fig. 1. Oncogenic fusion genes driving cholangiocarcinogenesis. Schematic diagram showing the discovery of fusion genes usingnext-generation sequencing (NGS); whole-genome sequencing (WGS) and RNA sequencing (RNA-seq). Tyrosine kinase gene fusionsresult in aberration of kinase signalling cascades and enhance CCA development. FGFR and ROS1 fusion genes served as the guidancefor targeted therapy in CCA.

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geographical and etiological origins, has yet to be fully characterized. While the identification of FGFRand ROS1 translocations may impact patient management, it is likely that further high-throughputgenomic profiling such as whole-genome sequencing or RNA-sequencing in larger cohorts of CCAmay reveal novel translocations of clinical relevance (Fig. 1).

Mutational landscape and associated dysregulated pathways in CCA

Genetic mutations are involved in the formation and progression of cancer, and therefore carrysignificant clinical implications from diagnosis to therapy. Mutations in well-known cancer driverssuch as TP53 and KRAS have been identified in many malignancies including CCA. Early studies of thetumour suppressor TP53, a master regulator of genomic stability, revealed a mutation rate of about 20%in CCA from all geographic areas including Asia, Europe and United States [58]. In TP53 mutant mice,addition of carbon tetrachloride (CCl4) caused the progression of epithelial hyperplasia of bile duct tomalignant ICC [59]. Activating KRAS mutations were found in both ICC and ECC, ranging in frequencyfrom 7% to 54% and were considered as early molecular events during progression from biliaryintraepithelial neoplasia to ICC [60e64]. Mutations of another proto-oncogene BRAF were found in upto 22% of ICC [63,65]. Taken together, genomic instability and RAS/RAF pathway may play importantroles in CCA tumorigenesis.

In recent years, high-throughput next-generation sequencing has enabled comprehensive muta-tional profiling of CCA, identifying novel mutated genes and providing new insights into the geneticbasis of CCA tumorigenesis [61,66,67]. The first study, using whole-exome sequencing of 8 Ov-relatedCCA, identified 206 somatic mutations in 187 genes. The prevalence of these mutations was validatedin additional 46 Ov-related CCA cases. Besides TP53 (44.4%) and KRAS (16.7%) described above, novelmutated CCA genes were identified: SMAD4 (16.7%), MLL3 (14.8%), RNF43 (9.3%), ROBO2 (9.3%), GNAS(9.3%), CDKN2A (5.6%) and PEG3 (5.6%) [61]. Interestingly, SMAD4 (16.7%) mutation frequency was

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similar to that of KRAS (16.7%) mutation. It has been shown to regulate the cell cycle mainly throughTGF-b signalling, suggesting a tumour suppressive role [68]. Inactivation of SMAD4 was previouslyfound in 35% of ICC and 50% of ECC [69]. Furthermore, both RNF43 and PEG3 are regulators of p53.RNF43, a RING domain E3 ubiquitin ligase, interacts with NEDL1 and p53, suppressing p53-mediatedapoptosis [70]. PEG3 is a maternally imprinted gene, and its encoded product induces apoptosisthrough interaction with Siah1a, an E3 ubiquitin ligase. Inhibition of PEG3 activity blocks p53-inducedapoptosis [71]. However, both RNF43 and PEG3 also play a role in the Wnt signalling pathway. PEG3inhibits Wnt signalling in human cells, and loss of PEG3 activates Wnt, leading to chromosomalinstability [72,73]. RNF43, on the other hand was shown to reduce Wnt signals by selectively ubiq-uinating frizzled receptors, targeting them for degradation [74]. Interestingly, activation of Wnt sig-nalling was previously observed in intrahepatic subtype of Ov-related CCA tumours based onoverexpression ofWnt3a,Wnt5a, andWnt7bmRNA [75], suggesting thatWnt signalling may be one ofthe key driver pathways in cholangiocarcinogenesis.

Another novel CCA-relatedmutated gene, ROBO2 receptor, has a similar structure to those of ROBO1and ROBO3, consisting of extracellular, transmembrane and cytoplasmic domains. The cytoplasmicdomain is inactive by itself. However, the Slit-Robo Rho GTPase-activating Protein 1 (srGAP1) can bindto the cytoplasmic domain of mammalian ROBO1, mediating Slit-dependent inactivation of the Rhofamily GTPase [76]. Thus, it is likely that loss of the cytoplasmic domain due to ROBO2 truncatingmutations may lead to a failure to switch off cellular signalling for growth and proliferation. ROBO2plays an important functional role in axon guidance during neuronal degeneration and sequencing ofpancreatic cancer genomes reveals aberrations in 20% of this cancer which is associated to Wnt sig-nalling [77]. Another tumour suppressor mutated CCA is CDKN2A, a negative regulator of cell cycleprogression that interacts with CDK4 and inhibit its kinase activity [78]. Previously, homozygous de-letions (5%) and loss of heterozygosity (20%) in the CDKN2A region have been found in CCAs [64],suggesting that inactivation of CDKN2A is a frequent event in CCA tumorigenesis.

A key group of genes that were found to be highly mutated in CCA through NGS studies arechromatin modifiers. These include MLL3, BAP1, ARID1A, PBRM1 and IDH. Notably, most of the tu-mours with MLL3 mutations did not habour TP53, KRAS or SMAD4 mutations, indicating that muta-tions of MLL3, a histone 3-lysine 4 (H3K4)-specific methyltransferase, may independently contributeto cholangiocarcinogenesis in this subset of tumours, probably through the downstream effects of itsassociated histone dysregulation (Fig. 2) [66,67]. BAP1 is a member of the ubiquitin C-terminal hy-drolases (UCH) subfamily of deubiquitylating enzymes. Complexed with ASXL1, BAP1 deubiquitinateshistone H2A [79]. Increased cell proliferation was observed after BAP1 knockdown whereas over-expression of wild-type BAP1 in non Ov-related CCA cell lines significantly suppressed cell prolifer-ation, suggesting the tumour suppressive role of this gene [66]. Interestingly, SWI/SNF complex,which is involved in nucleosome remodelling, appears to play an important role in chol-angiocarcinogenesis. It mediates ATP-dependent chromatin remodelling processes and exists in twoforms, BAF (BRG1-or hbrm-associated factors) and PBAF (polybromo-associated BAF) [80]. BothARID1A (a subunit of BAF complex) and PBRM1 (a subunit of PBAF complex) are frequently mutated

Fig. 2. The mutational landscape of Ov- and non-Ov-related CCA with difference of etiologies. Concurrent and mutually exclusivemutations are observed in the frequently mutated genes. Left column indicates genes validated in Ong CK. et al, 2012 and Chan-OnW. et al, 2013 [61,66] and top row indicates Ov-related status. Samples with or without mutations are labelled in colour or white,respectively.

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in CCA [81,82]. These genes have been previously found to be frequently mutated in clear cell ovariancarcinoma and renal cell carcinoma respectively [83e85]. Growing evidence indicate that thesecomplexes have a widespread role in tumour suppression, however the mechanisms by which mu-tations in these complexes drive tumorigenesis remain unclear. Silencing of ARID1A in CCA cell linesresulted in a significant increase in proliferation whereas overexpression of wild-type ARID1A led toretarded cell proliferation [66].

In recent years, IDHmutations in cancer have attracted significant attention and drugs targeting IDHhot-spot mutation are currently on clinical trial [86]. The mutant IDH protein converts a-ketoglutarate(a-KG) into an oncometabolite; 2-hydroxyglutarate, which competitively inhibits a-KG-dependentdioxygenase, including the TET family of 5-methylcytosine hydroxylases, leading to DNA methylationperturbation [87]. IDH1/2 mutations have been found in CCA, but the frequency of IDH mutationsvaries according to underlying etiology and geographical regions [66,67,87,88]. Furthermore, IDHmutations in CCA are associated with a hypermethylated phenotype, supporting the impact of IDH1/2mutations on global DNA methylation [66,87].

Collectively, genes affected by recurrent somatic alterations in CCA can be functionally grouped intothose involved in genomic stability, cell cycle control, Wnt signalling, cytokine signalling, TGF-b sig-nalling, MAPK signalling, AKT/PI3K signalling and epigenetic regulation (Fig. 3).

Other molecular pathways in CCA

Several other signalling pathways have also been proposed to play a role in cholangiocarcinogenesis(Fig. 3). Many of these pathways mediated oncogenic effects through their downstream effectors andmediators. Mitogen-activated protein kinases (MAPKs) signalling, for example, modulates oncogenicactivity with promoting proliferation, invasion, inflammation, and angiogenesis in cancers including

Fig. 3. An overview of common affected pathways in CCA. Pathways related to somatic mutations and overexpression are cate-gorized into eight pathways: genomic stability, cell cycle control, Wnt signalling, cytokine signalling, TGF-b signalling, MAPKssignalling, AKT/PI3K signalling and epigenetic regulation.

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CCA. p38delta has been proposed as a specific biomarker for CCA which is overexpressed at both theRNA and protein levels in CCA tumours compared to HCC or normal liver tissue [89]. Recently, ERBB2and MET oncogenes showed upregulated levels in CCA tumour and positive ERBB2 cases are highlycorrelated with lymph node metastasis [90]. Moreover, transfection of normal rat cholangiocytes withthe ERBB2 oncogene resulted in malignant neoplastic transformation with histological features ofhuman cholangiocarcinogenesis [91].

As described previously, inflammation is considered to be one of the key contributing factors in CCAdevelopment. IL-6 is an inflammatory cytokine released by tumour cells in response to external stimuli.Initially, IL-6 binds to the gp 130 receptor which then triggers the dimerization and the activation ofJAK kinases, subsequently leading to pSTAT3 activation. An integrative molecular study revealed thatpSTAT3 is upregulated in approximately 50% of ICC tumours [26]. Restoring SOCS-3 (upstream regu-lator of JAK/STAT) expression interrupts the activated signal from IL-6 through pSTAT in CCA cells andsensitizes the cells to apoptosis [39]. In a separate study, suppression of IL-6 mediated pSTAT3 reducedcolony forming ability and promoted cell-cycle arrest in CCA cell lines [92].

Genetically engineered mouse models

Several genetically engineered mouse models have reinforced the key roles played by thepathways described above in CCA initiation and progression (Table 1). Tissue-specific activation ofKRASG12D was sufficient for the development of invasive ICC. It promoted metastatic liver tumori-genesis that was significantly accelerated by the heterozygous and homozygous inactivation of p53[93]. The other models involved activation of two pathways or one pathway plus exposure to acarcinogen. Two independent studies reported that a combination of PTEN deletion with SMAD4inactivation or KRAS activation can provoke the development of CCA [94,95]. More recently, IDH andKRAS mutations, genetic alterations that co-exist in a subset of human ICC [87,96], cooperated todrive the expansion of liver progenitor cells and induced the development of ICC [17]. Finally, a CCAmouse model with PTEN and TP53 inactivation was generated using a new genetic engineeringtechnology, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associatedproteins) [97].

Table 1Genetically engineered animal models have been postulated the tumorigenesis and molecular pathogenesis in CCA.

Targeted pathways Genetic background Reference

1. TGF-b and PI3K signalling Liver specific- inactivation of SMAD4 and PTEN [95]2. p53 pathway Chronic CCl4 exposure in TP53-deficient mice [59]3. KRAS signalling Liver-specific activation of KRAS [93]4. KRAS signalling and p53 pathway Liver-specific activation of KRAS and deletion of p53 [93]5. KRAS/PI3K signalling Deletion of PTEN and KRAS activation within the

adult mouse biliary epithelium[94]

6. KRAS signalling and Epigenetic regulation IDH2 mutant and KRAS activation [17]7. PI3K signalling and p53 pathway CRISPR knockout of PTEN and p53 [97]

Summary

Recent advances in genomic profiling technologies have revealed novel genetic alterations in CCA,shedding light on the underlying molecular mechanisms of cholangiocarcinogenesis. Already theimportance of some of the molecular pathways associated with these genetic alterations have beenvalidated by mouse models. Furthermore, some of these alterations may have clinical implications,from diagnostic to therapeutic, although further studies involving larger cohort of samples are war-ranted. It is expected that even more genetic and epigenetic information related to CCA will begenerated in the near future which will open up greater opportunities for research on this deadlydisease.

Practice points

� CCA is a lethal malignancy with a poor prognosis. It is reported with 10e25% of all primary

liver cancers and its incidence is increasing worldwide. The incidence is known to be highest

in Southeast Asia especially in northeastern part of Thailand, Laos and Cambodia.

� Chronic inflammation caused by liver fluke infection and other diseases causing inflamed

bile duct such as PSC, hepatolithiasis are crucial risk factors of CCA. Recently, cirrhosis and

hepatitis B and C are identified as risk factors for ICC.

� New high-throughput genome-wide technologies and strategies have greatly increased our

understanding of the molecular mechanisms involved in CCA pathogenesis.

� Genomic and transcriptional analyses of CCA revealed distinct expression profiles, patterns

of chromosomal alterations, gene mutations and aberrant signalling pathways in different

etiology.

� The most common mutated genes in Ov-related CCA are TP53, SMAD4,MLL3, RNF43, PEG3

and ROBO2, whereas the epigenetic modulators BAP1, IDH1/2 and PBRM1 were more

frequently mutated in non-Ov group.

� Several potential biomarkers and therapeutic targets are currently being tested in key

pathways such as the inflammatory pathway, cell signalling pathways, growth factor sig-

nalling pathway and epigenetic regulation.

Research agenda

� Comprehensive analyses of the genomic and transcriptional alterations of CCA developed

with different etiology are necessary to define the precise underlying mechanisms.

� Generating of CCA animal models is essential for the development of new therapeutic

strategies and diagnostic tools.

� The focus on translating genomic and epigenetic studies into earlier diagnostic testing for

CCA, identification of promising target are yet to be fully characterized in a larger cohort to

gain more effective targeted therapies in CCA.

S. Kongpetch et al. / Best Practice & Research Clinical Gastroenterology 29 (2015) 233e244 241

Conflict of interest

None.

Acknowledgements

This work was supported in part by funding from the Singapore National Medical Research Council(NMRC/STAR/0006/2009), the Bronsveld Foundation (25560830), the Lee Foundation (Solexasequencing grant/26960760), the Tanoto Foundation (26961350), the Singapore National Cancer CentreResearch Fund (25560850), the Duke-NUS Graduate Medical School (R-913-200-070-263), the CancerScience Institute (R-713-006-011-271), Singapore and the Verdant Foundation, Hong Kong (N-918-041-003-001). The authors would like to thank Sabrina Noyes for assistance in submitting the manuscript.

References

[1] Vatanasapt V, Sriamporn S, Vatanasapt P. Cancer control in Thailand. Jpn J Clin Oncol 2002;32(Suppl.):S82e91.

S. Kongpetch et al. / Best Practice & Research Clinical Gastroenterology 29 (2015) 233e244242

[2] Shaib Y, El-Serag HB. The epidemiology of cholangiocarcinoma. Semin Liver Dis 2004;24:115e25.[3] Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver fluke induces cholangiocarcinoma. PLoS Med

2007;4:e201.[4] Huang MH, Chen CH, Yen CM, Yang JC, Yang CC, Yeh YH, et al. Relation of hepatolithiasis to helminthic infestation.

J Gastroenterol Hepatol 2005;20:141e6.[5] Zhou Y, Zhao Y, Li B, Huang J, Wu L, Xu D, et al. Hepatitis viruses infection and risk of intrahepatic cholangiocarcinoma:

evidence from a meta-analysis. BMC Cancer 2012;12:289.[6] Claessen MM, Vleggaar FP, Tytgat KM, Siersema PD, van Buuren HR. High lifetime risk of cancer in primary sclerosing

cholangitis. J Hepatol 2009;50:158e64.[7] Kobayashi M, Ikeda K, Saitoh S, Suzuki F, Tsubota A, Suzuki Y, et al. Incidence of primary cholangiocellular carcinoma of

the liver in japanese patients with hepatitis C virus-related cirrhosis. Cancer 2000;88:2471e7.*[8] Rizvi S, Gores GJ. Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology 2013;145:1215e29.[9] Shaib YH, El-Serag HB, Davila JA, Morgan R, McGlynn KA. Risk factors of intrahepatic cholangiocarcinoma in the United

States: a case-control study. Gastroenterology 2005;128:620e6.[10] Nakanuma Y, Sato Y, Harada K, Sasaki M, Xu J, Ikeda H. Pathological classification of intrahepatic cholangiocarcinoma

based on a new concept. World J Hepatol 2010;2:419e27.[11] Komuta M, Spee B, Vander Borght S, De Vos R, Verslype C, Aerts R, et al. Clinicopathological study on cholangiolocellular

carcinoma suggesting hepatic progenitor cell origin. Hepatology 2008;47:1544e56.[12] Tsuchiya A, Kamimura H, Tamura Y, Takamura M, Yamagiwa S, Suda T, et al. Hepatocellular carcinomawith progenitor cell

features distinguishable by the hepatic stem/progenitor cell marker NCAM. Cancer Lett 2011;309:95e103.[13] Holczbauer A, Factor VM, Andersen JB, Marquardt JU, Kleiner DE, Raggi C, et al. Modeling pathogenesis of primary liver

cancer in lineage-specific mouse cell types. Gastroenterology 2013;145:221e31.[14] Sekiya S, Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes. J Clin

Invest 2012;122:3914e8.[15] Fan B, Malato Y, Calvisi DF, Naqvi S, Razumilava N, Ribback S, et al. Cholangiocarcinomas can originate from hepatocytes in

mice. J Clin Invest 2012;122:2911e5.[16] Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, et al. Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling,

promotes development of human hepatocellular carcinoma. Gastroenterology 2011;140:1071e83.*[17] Saha SK, Parachoniak CA, Ghanta KS, Fitamant J, Ross KN, Najem MS, et al. Mutant IDH inhibits HNF-4alpha to block

hepatocyte differentiation and promote biliary cancer. Nature 2014;513:110e4.[18] Tamir S, Burney S, Tannenbaum SR. DNA damage by nitric oxide. Chem Res Toxicol 1996;9:821e7.[19] Wink DA, Grisham MB, Mitchell JB, Ford PC. Direct and indirect effects of nitric oxide in chemical reactions relevant to

biology. Methods Enzymol 1996;268:12e31.[20] Han C, Wu T. Cyclooxygenase-2-derived prostaglandin E2 promotes human cholangiocarcinoma cell growth and invasion

through EP1 receptor-mediated activation of the epidermal growth factor receptor and Akt. J Biol Chem 2005;280:24053e63.

[21] Pinlaor S, Hiraku Y, Ma N, Yongvanit P, Semba R, Oikawa S, et al. Mechanism of NO-mediated oxidative and nitrativeDNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis.Nitric Oxide 2004;11:175e83.

[22] Pinlaor S, Ma N, Hiraku Y, Yongvanit P, Semba R, Oikawa S, et al. Repeated infection with Opisthorchis viverrini inducesaccumulation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanine in the bile duct of hamsters via inducible nitricoxide synthase. Carcinogenesis 2004;25:1535e42.

*[23] Andersen JB, Thorgeirsson SS. Genetic profiling of intrahepatic cholangiocarcinoma. Curr Opin Gastroenterol 2012;28:266e72.

[24] Homayounfar K, Gunawan B, Cameron S, Haller F, Baumhoer D, Uecker S, et al. Pattern of chromosomal aberrations inprimary liver cancers identified by comparative genomic hybridization. Hum Pathol 2009;40:834e42.

[25] Koo SH, Ihm CH, Kwon KC, Park JW, Kim JM, Kong G. Genetic alterations in hepatocellular carcinoma and intrahepaticcholangiocarcinoma. Cancer Genet Cytogenet 2001;130:22e8.

[26] Sia D, Hoshida Y, Villanueva A, Roayaie S, Ferrer J, Tabak B. Integrative molecular analysis of intrahepatic chol-angiocarcinoma reveals 2 classes that have different outcomes. Gastroenterology 2013;144:829e40.

[27] McKay SC, Unger K, Pericleous S, Stamp G, Thomas G, Hutchins RR, et al. Array comparative genomic hybridizationidentifies novel potential therapeutic targets in cholangiocarcinoma. HPB Oxf 2011;13:309e19.

[28] Tsuda H, Satarug S, Bhudhisawasdi V, Kihana T, Sugimura T, Hirohashi S. Cholangiocarcinomas in Japanese and Thaipatients: difference in etiology and incidence of point mutation of the c-Ki-ras proto-oncogene. Mol Carcinog 1992;6:266e9.

[29] Yoshida S, Todoroki T, Ichikawa Y, Hanai S, Suzuki H, Hori M, et al. Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2genes in biliary tract cancers. Cancer Res 1995;55:2756e60.

[30] Kang MJ, Kim J, Jang JY, Park T, Lee KB, Kim SW. 22q11-q13 as a hot spot for prediction of disease-free survival in bile ductcancer: integrative analysis of copy number variations. Cancer Genet 2014;207:57e69.

[31] Limpaiboon T, Tapdara S, Jearanaikoon P, Sripa B, Bhudhisawasdi V. Prognostic significance of microsatellite alterations at1p36 in cholangiocarcinoma. World J Gastroenterol 2006;12:4377e82.

[32] Muenphon K, Limpaiboon T, Jearanaikoon P, Pairojkul C, Sripa B, Bhudhisawasdi V. Amplification of chromosome 21q22.3harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis. World JGastroenterol 2006;12:4143e8.

[33] Thanasai J, Limpaiboon T, Jearanaikoon P, Bhudhisawasdi V, Khuntikeo N, Sripa B, et al. Amplification of D22S283 as afavorable prognostic indicator in liver fluke related cholangiocarcinoma. World J Gastroenterol 2006;12:4338e44.

[34] Lee S, Kim WH, Jung HY, Yang MH, Kang GH. Aberrant CpG island methylation of multiple genes in intrahepatic chol-angiocarcinoma. Am J Pathol 2002;161:1015e22.

[35] Sandhu DS, Shire AM, Roberts LR. Epigenetic DNA hypermethylation in cholangiocarcinoma: potential roles in patho-genesis, diagnosis and identification of treatment targets. Liver Int 2008;28:12e27.

S. Kongpetch et al. / Best Practice & Research Clinical Gastroenterology 29 (2015) 233e244 243

[36] Tannapfel A, Sommerer F, Benicke M, Weinans L, Katalinic A, Geissler F, et al. Genetic and epigenetic alterations of theINK4a-ARF pathway in cholangiocarcinoma. J Pathol 2002;197:624e31.

[37] Yang B, House MG, Guo M, Herman JG, Clark DP. Promoter methylation profiles of tumor suppressor genes in intrahepaticand extrahepatic cholangiocarcinoma. Mod Pathol 2005;18:412e20.

[38] Xiaofang L, Kun T, Shaoping Y, Zaiqiu W, Hailong S. Correlation between promoter methylation of p14(ARF), TMS1/ASC,and DAPK, and p53 mutation with prognosis in cholangiocarcinoma. World J Surg Oncol 2012;10:5.

[39] Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S, et al. Sustained IL-6/STAT-3 signaling in chol-angiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology 2007;132:384e96.

[40] Andresen K, Boberg KM, Vedeld HM, Honne H, Hektoen M,Wadsworth CA, et al. Novel target genes and a valid biomarkerpanel identified for cholangiocarcinoma. Epigenetics 2012;7:1249e57.

[41] Goeppert B, Konermann C, Schmidt CR, Bogatyrova O, Geiselhart L, Ernst C, et al. Global alterations of DNA methylation incholangiocarcinoma target the Wnt signaling pathway. Hepatology 2014;59:544e54.

[42] Maemura K, Natsugoe S, Takao S. Molecular mechanism of cholangiocarcinoma carcinogenesis. J Hepatobiliary PancreatSci 2014;21:754e60.

[43] Zhang J, Han C, Wu T. MicroRNA-26a promotes cholangiocarcinoma growth by activating beta-catenin. Gastroenterology2012;143:246e56. e8.

[44] Kawahigashi Y, Mishima T, Mizuguchi Y, Arima Y, Yokomuro S, Kanda T, et al. MicroRNA profiling of human intrahepaticcholangiocarcinoma cell lines reveals biliary epithelial cell-specific microRNAs. J Nippon Med Sch 2009;76:188e97.

[45] Selaru FM, Olaru AV, Kan T, David S, Cheng Y, Mori Y, et al. MicroRNA-21 is overexpressed in human cholangiocarcinomaand regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3. Hepatology 2009;49:1595e601.

[46] Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT, et al. Involvement of human micro-RNA in growth andresponse to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology 2006;130:2113e29.

[47] Razumilava N, Bronk SF, Smoot RL, Fingas CD, Werneburg NW, Roberts LR, et al. miR-25 targets TNF-related apoptosisinducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma. Hepatology 2012;55:465e75.

[48] Meng F, Henson R, Wehbe-Janek H, Smith H, Ueno Y, Patel T. The MicroRNA let-7a modulates interleukin-6-dependentSTAT-3 survival signaling in malignant human cholangiocytes. J Biol Chem 2007;282:8256e64.

[49] Zhong XY, Yu JH, Zhang WG, Wang ZD, Dong Q, Tai S, et al. MicroRNA-421 functions as an oncogenic miRNA in biliarytract cancer through down-regulating farnesoid X receptor expression. Gene 2012;493:44e51.

[50] Lupski JR, Stankiewicz P. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoSGenet 2005;1:e49.

[51] Charest A, Lane K, McMahon K, Park J, Preisinger E, Conroy H, et al. Fusion of FIG to the receptor tyrosine kinase ROS in aglioblastoma with an interstitial del(6)(q21q21). Genes Chromosom Cancer 2003;37:58e71.

[52] Shaw AT, Hsu PP, Awad MM, Engelman JA. Tyrosine kinase gene rearrangements in epithelial malignancies. Nat RevCancer 2013;13:772e87.

[53] Gu TL, Deng X, Huang F, Tucker M, Crosby K, Rimkunas V, et al. Survey of tyrosine kinase signaling reveals ROS kinasefusions in human cholangiocarcinoma. PLoS One 2011;6:e15640.

[54] Saborowski A, Saborowski M, Davare MA, Druker BJ, Klimstra DS, Lowe SW. Mouse model of intrahepatic chol-angiocarcinoma validates FIG-ROS as a potent fusion oncogene and therapeutic target. Proc Natl Acad Sci U S A 2013;110:19513e8.

*[55] Arai Y, Totoki Y, Hosoda F, Shirota T, Hama N, Nakamura H, et al. Fibroblast growth factor receptor 2 tyrosine kinasefusions define a unique molecular subtype of cholangiocarcinoma. Hepatology 2014;59:1427e34.

[56] Borad MJ, Champion MD, Egan JB, LiangWS, Fonseca R, Bryce AH, et al. Integrated genomic characterization reveals novel,therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma. PLoSGenet 2014;10:e1004135.

*[57] Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, et al. Identification of targetable FGFR gene fusions indiverse cancers. Cancer Discov 2013;3:636e47.

[58] Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. Cholangiocarcinoma. Lancet 2005;366:1303e14.[59] Farazi PA, Zeisberg M, Glickman J, Zhang Y, Kalluri R, DePinho RA. Chronic bile duct injury associated with fibrotic matrix

microenvironment provokes cholangiocarcinoma in p53-deficient mice. Cancer Res 2006;66:6622e7.[60] Hsu M, Sasaki M, Igarashi S, Sato Y, Nakanuma Y. KRAS and GNAS mutations and p53 overexpression in biliary intra-

epithelial neoplasia and intrahepatic cholangiocarcinomas. Cancer 2013;119:1669e74.*[61] Ong CK, Subimerb C, Pairojkul C, Wongkham S, Cutcutache I, Yu W, et al. Exome sequencing of liver fluke-associated

cholangiocarcinoma. Nat Genet 2012;44:690e3.[62] Rashid A, Ueki T, Gao YT, Houlihan PS, Wallace C, Wang BS, et al. K-ras mutation, p53 overexpression, and microsatellite

instability in biliary tract cancers: a population-based study in China. Clin Cancer Res 2002;8:3156e63.[63] Robertson S, Hyder O, Dodson R, Nayar SK, Poling J, Beierl K, et al. The frequency of KRAS and BRAF mutations in

intrahepatic cholangiocarcinomas and their correlation with clinical outcome. Hum Pathol 2013;44:2768e73.[64] Tannapfel A, Benicke M, Katalinic A, Uhlmann D, Kockerling F, Hauss J, et al. Frequency of p16(INK4A) alterations and K-

ras mutations in intrahepatic cholangiocarcinoma of the liver. Gut 2000;47:721e7.[65] Tannapfel A, Sommerer F, Benicke M, Katalinic A, Uhlmann D, Witzigmann H, et al. Mutations of the BRAF gene in

cholangiocarcinoma but not in hepatocellular carcinoma. Gut 2003;52:706e12.*[66] Chan-On W, Nairismagi ML, Ong CK, Lim WK, Dima S, Pairojkul C, et al. Exome sequencing identifies distinct mutational

patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet 2013;45:1474e8.*[67] Jiao Y, Pawlik TM, Anders RA, Selaru FM, Streppel MM, Lucas DJ, et al. Exome sequencing identifies frequent inactivating

mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 2013;45:1470e3.[68] Lagna G, Hata A, Hemmati-Brivanlou A, Massague J. Partnership between DPC4 and SMAD proteins in TGF-beta signalling

pathways. Nature 1996;383:832e6.[69] Chuang SC, Lee KT, Tsai KB, Sheen PC, Nagai E, Mizumoto K, et al. Immunohistochemical study of DPC4 and p53 proteins

in gallbladder and bile duct cancers. World J Surg 2004;28:995e1000.

S. Kongpetch et al. / Best Practice & Research Clinical Gastroenterology 29 (2015) 233e244244

[70] Shinada K, Tsukiyama T, Sho T, Okumura F, Asaka M, Hatakeyama S. RNF43 interacts with NEDL1 and regulates p53-mediated transcription. Biochem Biophys Res Commun 2011;404:143e7.

[71] Deng Y, Wu X. Peg3/Pw1 promotes p53-mediated apoptosis by inducing Bax translocation from cytosol to mitochondria.Proc Natl Acad Sci U S A 2000;97:12050e5.

[72] Aoki K, Aoki M, Sugai M, Harada N, Miyoshi H, Tsukamoto T, et al. Chromosomal instability by beta-catenin/TCF tran-scription in APC or beta-catenin mutant cells. Oncogene 2007;26:3511e20.

[73] Jiang X, Yu Y, Yang HW, Agar NY, Frado L, Johnson MD. The imprinted gene PEG3 inhibits Wnt signaling and regulatesglioma growth. J Biol Chem 2010;285:8472e80.

[74] Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, et al. Tumour suppressor RNF43 is a stem-cell E3 ligasethat induces endocytosis of Wnt receptors. Nature 2012;488:665e9.

[75] Loilome W, Bungkanjana P, Techasen A, Namwat N, Yongvanit P, Puapairoj A, et al. Activated macrophages promote Wnt/beta-catenin signaling in cholangiocarcinoma cells. Tumour Biol 2014;35:5357e67.

[76] Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, et al. Signal transduction in neuronal migration: roles of GTPaseactivating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 2001;107:209e21.

[77] Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, et al. Pancreatic cancer genomes revealaberrations in axon guidance pathway genes. Nature 2012;491:399e405.

[78] Huschtscha LI, Reddel RR. p16(INK4a) and the control of cellular proliferative life span. Carcinogenesis 1999;20:921e6.[79] Scheuermann JC, de Ayala Alonso AG, Oktaba K, Ly-Hartig N, McGinty RK, Fraterman S, et al. Histone H2A deubiquitinase

activity of the polycomb repressive complex PR-DUB. Nature 2010;465:243e7.[80] Nie Z, Yan Z, Chen EH, Sechi S, Ling C, Zhou S, et al. Novel SWI/SNF chromatin-remodeling complexes contain a mixed-

lineage leukemia chromosomal translocation partner. Mol Cell Biol 2003;23:2942e52.[81] Kwon H, Imbalzano AN, Khavari PA, Kingston RE, Green MR. Nucleosome disruption and enhancement of activator

binding by a human SW1/SNF complex. Nature 1994;370:477e81.[82] Wang W, Xue Y, Zhou S, Kuo A, Cairns BR, Crabtree GR. Diversity and specialization of mammalian SWI/SNF complexes.

Genes Dev 1996;10:2117e30.[83] Jones S, Wang TL, Shih Ie M, Mao TL, Nakayama K, Roden R, et al. Frequent mutations of chromatin remodeling gene

ARID1A in ovarian clear cell carcinoma. Science 2010;330:228e31.[84] Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, et al. Exome sequencing identifies frequent mutation of the SWI/

SNF complex gene PBRM1 in renal carcinoma. Nature 2011;469:539e42.[85] Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, et al. ARID1A mutations in endometriosis-associated ovarian

carcinomas. N Engl J Med 2010;363:1532e43.[86] Okosun J, Packham G, Fitzgibbon J. Investigational epigenetically targeted drugs in early phase trials for the treatment of

haematological malignancies. Expert Opin Investig Drugs 2014;23:1321e32.*[87] Wang P, Dong Q, Zhang C, Kuan PF, Liu Y, JeckWR, et al. Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in

intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas. Oncogene 2013;32:3091e100.[88] Borger DR, Tanabe KK, Fan KC, Lopez HU, Fantin VR, Straley KS, et al. Frequent mutation of isocitrate dehydrogenase (IDH)

1 and IDH2 in cholangiocarcinoma identified through broad-based tumor genotyping. Oncologist 2012;17:72e9.[89] Tan FL, Ooi A, Huang D, Wong JC, Qian CN, Chao C, et al. p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma

and its involvement in cell motility and invasion. Int J Cancer 2010;126:2353e61.[90] Aishima SI, Taguchi KI, Sugimachi K, Shimada M, Tsuneyoshi M. c-erbB-2 and c-Met expression relates to chol-

angiocarcinogenesis and progression of intrahepatic cholangiocarcinoma. Histopathology 2002;40:269e78.[91] Lai GH, Zhang Z, Shen XN, Ward DJ, Dewitt JL, Holt SE, et al. erbB-2/neu transformed rat cholangiocytes recapitulate key

cellular and molecular features of human bile duct cancer. Gastroenterology 2005;129:2047e57.[92] Aneknan P, Kukongviriyapan V, Prawan A, Kongpetch S, Sripa B, Senggunprai L. Luteolin arrests cell cycling, induces

apoptosis and inhibits the JAK/STAT3 pathway in human cholangiocarcinoma cells. Asian Pac J Cancer Prev 2014;15:5071e6.

[93] O'Dell MR, Huang JL, Whitney-Miller CL, Deshpande V, Rothberg P, Grose V, et al. Kras(G12D) and p53 mutation causeprimary intrahepatic cholangiocarcinoma. Cancer Res 2012;72:1557e67.

[94] Marsh V, Davies EJ, Williams GT, Clarke AR. PTEN loss and KRAS activation cooperate in murine biliary tract malignancies.J Pathol 2013;230:165e73.

[95] Xu X, Kobayashi S, Qiao W, Li C, Xiao C, Radaeva S, et al. Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice. J Clin Invest 2006;116:1843e52.

[96] Voss JS, Holtegaard LM, Kerr SE, Fritcher EG, Roberts LR, Gores GJ, et al. Molecular profiling of cholangiocarcinoma showspotential for targeted therapy treatment decisions. Hum Pathol 2013;44:1216e22.

*[97] XueW, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, et al. CRISPR-mediated direct mutation of cancer genesin the mouse liver. Nature 2014;514:380e4.