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

Contents lists available at ScienceDirect

Best Practice & Research Clinical

Gastroenterology

Pathogenesis of cholangiocarcinoma: From

genetics 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,
Singapore
b
Department of Pharmacology, Faculty of Medicine and Liver Fluke and Cholangiocarcinoma Research
Center, Khon Kaen University, Khon Kaen, Thailand
c
Division of Cancer and Stem Cell Biology, Duke-National University of Singapore (NUS) Graduate Medical
School, Singapore
d
Centre for Computational Biology, Duke-NUS Graduate Medical School, Singapore
e
Genome Institute of Singapore, Singapore
f
Cancer Science Institute of Singapore, National University of Singapore, Singapore

a b s t r a c t
Keywords:
Cholangiocarcinoma
Molecular pathogenesis
Genetic alteration
Chromatin

Cholangiocarcinoma (CCA) is a malignant tumour of bile duct

epithelial cells with dismal prognosis and rising incidence. Chronic
inammation resulting from liver uke infection, hepatitis and
other inammatory bowel diseases is a major contributing factor
to cholangiocarcinogenesis, likely through accumulation of serial
genetic and epigenetic alterations resulting in aberration of oncogenes and tumour suppressors. Recent studies making use of
advances in high-throughput genomics have revealed the genetic
landscape of CCA, greatly increasing our understanding of its underlying biology. A series of highly recurrent mutations in genes

* Corresponding author. Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore,
Singapore. Tel.: 65 66 011324.
E-mail addresses: sarinya.kongpetch@nccs.com.sg, sarinyako@kku.ac.th (S. Kongpetch), apinya.jusakul@duke-nus.edu.sg
(A. Jusakul), cmrock@nccs.com.sg (C.K. Ong), wengkhong.lim@duke-nus.edu.sg (W.K. Lim), steve.rozen@duke-nus.edu.sg
(S.G. Rozen), gmstanp@duke-nus.edu.sg (P. Tan), teh.bin.tean@singhealth.com.sg (B.T. Teh).

http://dx.doi.org/10.1016/j.bpg.2015.02.002
1521-6918/ 2015 Elsevier Ltd. All rights reserved.

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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 signalling
pathways and chromatin dynamics, have led to investigations of
their roles, through molecular to mouse modelling studies, in
cholangiocarcinogenesis. This review focuses on the landscape
genetic alterations in CCA and its functional relevance to the formation 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 all
primary liver cancers diagnosed worldwide. Its incidence is highest in northeastern Thailand, bordering
Laos and Cambodia, with very high age-standardized incidence rates (ASRs) of 84.6 and 36.8 per
100,000 in males and females, respectively [1]. This is in contrast to ASRs of less than 1.5 per 100,000 in
Western countries [2]. Several risk factors for CCA are related to geography and etiology. For instance,
infestation of liver ukes such as Opisthorchis viverrini (Ov) and Clonorchis sinensis has been associated
with 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 uke
infestation [4]. Cirrhosis, hepatitis B (HBV) and hepatitis C viral (HCV) infection among other risk factors
identied from meta-analysis [5]. In contrast, primary sclerosing cholangitis (PSC) is the most common
risk factor of CCA in the Western countries. The well-established association between PSC and CCA is
marked by chronic inammation, resulting in liver injury and likely proliferation of the progenitor cells
[6]. Other potential contributing factors to ICC include HIV infection, inammatory bowel disease independent 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 inammation in CCA.
In this review, we focus on recurrent alterations in the genetic landscape of CCA. The spectrum and
frequency of these alterations, including those identied from recent whole-exome sequencing
studies, indicate the possible involvement of their associated molecular pathways in CCA. This is
further substantiated by in vitro and in vivo studies. The results from these experiments, especially the
latter, have potential clinical implications as they point to the importance of targeting specic altered
pathways 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 be
categorized into two common groups by anatomical location; intrahepatic (ICC) and extrahepatic
cholangiocarcinoma (ECC). ICC refers to tumours arising from the large and small bile ducts within the
liver. ECC, on the other hand, refers to bile duct tumours arising outside the liver, that can be further
divided into perihilar and distal CCAs, separated by the junction of cystic and common bile ducts [8].
The traditional classication of ICC includes well, moderately and poorly differentiated adenocarcinomas. Recently, there is a new pathological concept to classify ICC into conventional ICC, bile ductular
ICC, intraductal neoplasms and rare variants (combined hepatocellular CCA, undifferentiated type,
squamous/adenosquamous type) [10]. Interestingly, a marker of hepatic progenitor cells has been
detected in the bile ductular and combined hepatocellular CCA types, suggesting these may have
originated from hepatic progenitor cells [11,12]. Recent studies also propose that rather than being of
single cellular origin, CCA may have developed from a combination of cholangiocyte, the peribiliary
gland around bile duct, hepatic progenitor cell or hepatocytes [8]. Mouse models have shown that
transformed hepatocytes, hepatoblasts, and hepatic progenitor cells are capable of producing a broad
spectrum of liver malignancies ranging from CCA to hepatocellular carcinoma (HCC) [13]. These studies

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235

suggest that cholangiocytes alone may not be sufcient for CCA carcinogenesis, and that this process
may involve the transformation of multiple cell types. In one study, Sekiya et al reported that hepatocytes can transform into biliary cells through the Notch pathway, leading to ICC formation [14]. In
another, 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 overexpression alone may not be sufcient for ICC formation [16]. More recently, it was shown that mice
engineered to express both mutant IDH2 and KRAS in the adult liver displayed phenotypes such as the
expansion of liver progenitor cells, development of premalignant biliary lesions, and nally progression to metastatic ICC [17].
Molecular and cellular pathogenesis
As alluded to above, chronic infection and inammation in the bile ducts play an important role in
cholangiocarcinogenesis. Inammation causes the release of proinammatory cytokines leading to
induction of nitric oxide synthase (iNOS), a generator of nitric oxide (NO) in cholangiocytes. NO produced in infected and inamed tissues has been postulated to contribute to epithelial cell carcinogenesis by causing damage to DNA and proteins [18]. NO can also directly oxidize DNA, resulting in
mutagenic changes [19] and stimulates cyclooxygenase-2 (COX-2) expression promoting cholangiocyte
growth via activation of growth factors such as EGFR, MAPK, and IL-6 [20]. In the hamster CCA model,
chronic inammation triggered by repeated Ov infection was reported to mediate iNOS-dependent
DNA damage in intrahepatic bile duct epithelium and inammatory cells, and the combination of Ov
infection and exposure of nitrosamine led to development of CCA [21,22]. Obviously, advances in
cancer genomics as a result of more effective and high-throughput proling technologies have allowed
characterization of the genetic alterations, including their spectrum and frequency, in CCA associated
with different etiological factors.
Chromosomal changes
Several studies have described chromosomal aberrations in CCA. A meta-analysis of comparative
genomic hybridization studies identied common chromosomal gains at 1q, 5p, 7p, 8q, 17q and 20q as
well as losses at 1p, 4q, 8p, 9p, 17p and 18q [23]. Patterns of genomic changes reect differences in
relation to ethnicity and etiology. Tumour samples from Asian countries reveal common patterns of
gains in chromosomes 5p, 6p, 7p, 8q, 11q, 13q, 17q, and 20q and losses at 4q, 6q, 8p, 10p, 17q, 18q, and
22q [24,25], whereas karyotyping of European CCA cases showed greater diversity. The only regions
shared 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 CCA
and HCC, implying that there may be a close relationship between these two cancer types [23].
In a separate study, signicant gains of 2p, 5p, 22q and signicant losses of 8q, 10q, 11p, and 18q
were observed in CCA and these chromosomal regions contained approximately 153 genes, some of
which may serve as oncogenes or tumour suppressor genes including those involved in JAK-STAT and
MAPK pathways. Other studies also showed gains and losses of chromosomal regions containing
cancer-driving genes such as ERBB2/HER2 on 17q, MAP2K2/MEKs on 19p, EGFR on 7p12, PDGFA on
7p22, CDKN2A on 9p21 and TP53 on 17p13 [27e29]. Interestingly, copy number gains at 5p15.33 and
22q13.33 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 and
losses of 1p36, 9p21, 17q13 and 22q12 [31e33].
Aberrant epigenetic landscape
Epigenetic dysregulation including histone modication and DNA methylation has been implicated
in the pathogenesis of many cancers including CCA. In tumours, the aberrant DNA methylation occurs
at the 50 methylcytosine (5-mc) in CpG rich area in the promotor region of tumour suppressor genes
leading 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, TP53 mutation with hypermethylated promoter of p14ARF, DAPK, and/or ASC appeared to
contribute to more aggressive CCA and shorter survival [38].
Epigenetic changes in the genes linked to cytokine and other signalling pathways have also been
implicated in CCA. For example, the promoter of SOCS3, which is the upstream regulator of JAK/STAT
cytokine 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 poor
prognosis [41]. In the future, epigenomic proling of CCA including histone marks, promoters and
enhancers may further shed light on CCA tumorigenesis and progression.
microRNAs (miRNAs) dysregulation
miRNAs are small noncoding RNA that are approximately 20e22 nucleotides in length. They
negatively regulated target gene expression by binding to 30 UTR sites, leading to translational inhibition as well as mRNA degradation. Dysregulated miRNAs have been implicated in cancer development including CCA tumorigenesis. These miRNAs regulating oncogenes (onco-miRNAs) are involved
in biological processes, from cell cycle, apoptosis to cancer metabolism at the post-transcriptional level
[42,43]. A comprehensive proling of miRNA in CCA cell lines (HUCCT1 and MEC) revealed biliary
epithelial cell-specic miRNAs, i.e., miR22, miR125a, miR127, miR199a, miR199a*, miR214, miR376a
and miR424, which are downregulated in these lines [44]. In a separate study, miR21 was found to be
upregulated in ICC compared to normal epithelial bile duct tissue. Inhibition of miR21 was shown to
increase protein expression of PDCD4 and TIMP3 which are the inhibitors of program-cell death and
metastasis, respectively [45]. Moreover, miR21 was shown to stimulate CCA cell growth and resistance
to chemotherapy by inhibiting PTEN, a tumour suppressor [46]. Other studies have shown that miR25
has an anti-apoptotic effect in CCA via inhibiting the death receptor, TRAIL (TNF-related apoptosisinducing ligand) [47] whereas miR26a, acting on its downstream GSK-3b, could mediate intracellular
accumulation of b-catenin, promoting proliferation and colony formation in cholangiocarcinoma [43].
Other dysregulated miRNAs in CCA include miRlet7a (activator of STAT3 signalling pathway) and
miR421 (suppressor of tumour suppressor gene FXR), and these have been shown to regulate cell
proliferation, colony formation and migration [48,49].
Structural variation driving cholangiocarcinogenesis
There is emerging evidence of the involvement of novel genomic rearrangements in epithelial
cancers such as CCA. These genomic rearrangements include gene amplications, chromosomal
translocations, inversions and deletions. They may represent polymorphisms that are neutral in
function, or convey phenotypes such as changing the copy number, disrupting genes and creating
fusion genes [50]. Gene fusions resulting from chromosomal rearrangements are one of the most
common events, often considered as onco-fusion proteins in cancer development [51,52]. Many fusion
kinases with active kinase domains have been associated with tumour initiation via activation of
downstream kinases leading to progressive phenotypes in cancer (Fig. 1). Tyrosine kinase gene fusions
such as ROS and FGFR gene fusions with intact kinase domains were identied in various cancer types.
ROS1 translocation was reported in 9% of CCA patients [53]. Later on, a mouse model habouring FIGeROS1 fusion gene that eventually promoted ICC development was generated [54]. More recently, RNA
sequencing studies have reported FGFR2 gene fusions in CCA tumours [55e57]. Importantly, such
fusion proteins may serve as potential therapeutic targets for FGFR inhibitors. One study reported
FGFR2-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 increased
cell proliferation. In vitro and in vivo studies demonstrated increasing sensitivity to FGFR inhibitors in
mouse broblast and bladder cancer cell lines that haboured FGFR fusions [55,57]. Finally, treatment
with FGFR inhibitors such as pazopanib and ponatinib in patients has shown improved clinical responses 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

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237

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

geographical and etiological origins, has yet to be fully characterized. While the identication of FGFR
and ROS1 translocations may impact patient management, it is likely that further high-throughput
genomic proling such as whole-genome sequencing or RNA-sequencing in larger cohorts of CCA
may 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 carry
signicant clinical implications from diagnosis to therapy. Mutations in well-known cancer drivers
such as TP53 and KRAS have been identied in many malignancies including CCA. Early studies of the
tumour 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 to
malignant ICC [59]. Activating KRAS mutations were found in both ICC and ECC, ranging in frequency
from 7% to 54% and were considered as early molecular events during progression from biliary
intraepithelial neoplasia to ICC [60e64]. Mutations of another proto-oncogene BRAF were found in up
to 22% of ICC [63,65]. Taken together, genomic instability and RAS/RAF pathway may play important
roles in CCA tumorigenesis.
In recent years, high-throughput next-generation sequencing has enabled comprehensive mutational proling of CCA, identifying novel mutated genes and providing new insights into the genetic
basis of CCA tumorigenesis [61,66,67]. The rst study, using whole-exome sequencing of 8 Ov-related
CCA, identied 206 somatic mutations in 187 genes. The prevalence of these mutations was validated
in additional 46 Ov-related CCA cases. Besides TP53 (44.4%) and KRAS (16.7%) described above, novel
mutated CCA genes were identied: 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 through
TGF-b signalling, suggesting a tumour suppressive role [68]. Inactivation of SMAD4 was previously
found 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-mediated
apoptosis [70]. PEG3 is a maternally imprinted gene, and its encoded product induces apoptosis
through interaction with Siah1a, an E3 ubiquitin ligase. Inhibition of PEG3 activity blocks p53-induced
apoptosis [71]. However, both RNF43 and PEG3 also play a role in the Wnt signalling pathway. PEG3
inhibits Wnt signalling in human cells, and loss of PEG3 activates Wnt, leading to chromosomal
instability [72,73]. RNF43, on the other hand was shown to reduce Wnt signals by selectively ubiquinating frizzled receptors, targeting them for degradation [74]. Interestingly, activation of Wnt signalling was previously observed in intrahepatic subtype of Ov-related CCA tumours based on
overexpression of Wnt3a, Wnt5a, and Wnt7b mRNA [75], suggesting that Wnt signalling may be one of
the key driver pathways in cholangiocarcinogenesis.
Another novel CCA-related mutated gene, ROBO2 receptor, has a similar structure to those of ROBO1
and ROBO3, consisting of extracellular, transmembrane and cytoplasmic domains. The cytoplasmic
domain is inactive by itself. However, the Slit-Robo Rho GTPase-activating Protein 1 (srGAP1) can bind
to the cytoplasmic domain of mammalian ROBO1, mediating Slit-dependent inactivation of the Rho
family GTPase [76]. Thus, it is likely that loss of the cytoplasmic domain due to ROBO2 truncating
mutations may lead to a failure to switch off cellular signalling for growth and proliferation. ROBO2
plays an important functional role in axon guidance during neuronal degeneration and sequencing of
pancreatic cancer genomes reveals aberrations in 20% of this cancer which is associated to Wnt signalling [77]. Another tumour suppressor mutated CCA is CDKN2A, a negative regulator of cell cycle
progression that interacts with CDK4 and inhibit its kinase activity [78]. Previously, homozygous deletions (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 are
chromatin modiers. These include MLL3, BAP1, ARID1A, PBRM1 and IDH. Notably, most of the tumours with MLL3 mutations did not habour TP53, KRAS or SMAD4 mutations, indicating that mutations of MLL3, a histone 3-lysine 4 (H3K4)-specic methyltransferase, may independently contribute
to cholangiocarcinogenesis in this subset of tumours, probably through the downstream effects of its
associated histone dysregulation (Fig. 2) [66,67]. BAP1 is a member of the ubiquitin C-terminal hydrolases (UCH) subfamily of deubiquitylating enzymes. Complexed with ASXL1, BAP1 deubiquitinates
histone H2A [79]. Increased cell proliferation was observed after BAP1 knockdown whereas overexpression of wild-type BAP1 in non Ov-related CCA cell lines signicantly suppressed cell proliferation, 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 cholangiocarcinogenesis. It mediates ATP-dependent chromatin remodelling processes and exists in two
forms, BAF (BRG1-or hbrm-associated factors) and PBAF (polybromo-associated BAF) [80]. Both
ARID1A (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 exclusive
mutations are observed in the frequently mutated genes. Left column indicates genes validated in Ong CK. et al, 2012 and Chan-On
W. 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 ovarian
carcinoma and renal cell carcinoma respectively [83e85]. Growing evidence indicate that these
complexes have a widespread role in tumour suppression, however the mechanisms by which mutations in these complexes drive tumorigenesis remain unclear. Silencing of ARID1A in CCA cell lines
resulted in a signicant increase in proliferation whereas overexpression of wild-type ARID1A led to
retarded cell proliferation [66].
In recent years, IDH mutations in cancer have attracted signicant attention and drugs targeting IDH
hot-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-dependent
dioxygenase, including the TET family of 5-methylcytosine hydroxylases, leading to DNA methylation
perturbation [87]. IDH1/2 mutations have been found in CCA, but the frequency of IDH mutations
varies according to underlying etiology and geographical regions [66,67,87,88]. Furthermore, IDH
mutations in CCA are associated with a hypermethylated phenotype, supporting the impact of IDH1/2
mutations on global DNA methylation [66,87].
Collectively, genes affected by recurrent somatic alterations in CCA can be functionally grouped into
those involved in genomic stability, cell cycle control, Wnt signalling, cytokine signalling, TGF-b signalling, 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 and
mediators. Mitogen-activated protein kinases (MAPKs) signalling, for example, modulates oncogenic
activity with promoting proliferation, invasion, inammation, and angiogenesis in cancers including

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

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CCA. p38delta has been proposed as a specic biomarker for CCA which is overexpressed at both the
RNA and protein levels in CCA tumours compared to HCC or normal liver tissue [89]. Recently, ERBB2
and MET oncogenes showed upregulated levels in CCA tumour and positive ERBB2 cases are highly
correlated with lymph node metastasis [90]. Moreover, transfection of normal rat cholangiocytes with
the ERBB2 oncogene resulted in malignant neoplastic transformation with histological features of
human cholangiocarcinogenesis [91].
As described previously, inammation is considered to be one of the key contributing factors in CCA
development. IL-6 is an inammatory 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 of
JAK kinases, subsequently leading to pSTAT3 activation. An integrative molecular study revealed that
pSTAT3 is upregulated in approximately 50% of ICC tumours [26]. Restoring SOCS-3 (upstream regulator of JAK/STAT) expression interrupts the activated signal from IL-6 through pSTAT in CCA cells and
sensitizes the cells to apoptosis [39]. In a separate study, suppression of IL-6 mediated pSTAT3 reduced
colony 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 the
pathways described above in CCA initiation and progression (Table 1). Tissue-specic activation of
KRASG12D was sufcient for the development of invasive ICC. It promoted metastatic liver tumorigenesis that was signicantly accelerated by the heterozygous and homozygous inactivation of p53
[93]. The other models involved activation of two pathways or one pathway plus exposure to a
carcinogen. Two independent studies reported that a combination of PTEN deletion with SMAD4
inactivation or KRAS activation can provoke the development of CCA [94,95]. More recently, IDH and
KRAS mutations, genetic alterations that co-exist in a subset of human ICC [87,96], cooperated to
drive the expansion of liver progenitor cells and induced the development of ICC [17]. Finally, a CCA
mouse model with PTEN and TP53 inactivation was generated using a new genetic engineering
technology, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated
proteins) [97].

Table 1
Genetically engineered animal models have been postulated the tumorigenesis and molecular pathogenesis in CCA.
Targeted pathways
1.
2.
3.
4.
5.

TGF-b and PI3K signalling

p53 pathway
KRAS signalling
KRAS signalling and p53 pathway
KRAS/PI3K signalling

6. KRAS signalling and Epigenetic regulation

7. PI3K signalling and p53 pathway

Genetic background

Reference

Liver specic- inactivation of SMAD4 and PTEN

Chronic CCl4 exposure in TP53-decient mice
Liver-specic activation of KRAS
Liver-specic activation of KRAS and deletion of p53
Deletion of PTEN and KRAS activation within the
adult mouse biliary epithelium
IDH2 mutant and KRAS activation
CRISPR knockout of PTEN and p53

[95]
[59]
[93]
[93]
[94]
[17]
[97]

Summary
Recent advances in genomic proling technologies have revealed novel genetic alterations in CCA,
shedding light on the underlying molecular mechanisms of cholangiocarcinogenesis. Already the
importance of some of the molecular pathways associated with these genetic alterations have been
validated 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 warranted. It is expected that even more genetic and epigenetic information related to CCA will be
generated in the near future which will open up greater opportunities for research on this deadly
disease.

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241

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 signalling 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.

Conict 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 (Solexa
sequencing grant/26960760), the Tanoto Foundation (26961350), the Singapore National Cancer Centre
Research Fund (25560850), the Duke-NUS Graduate Medical School (R-913-200-070-263), the Cancer
Science Institute (R-713-006-011-271), Singapore and the Verdant Foundation, Hong Kong (N-918-041003-001). The authors would like to thank Sabrina Noyes for assistance in submitting the manuscript.
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