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

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Best Practice & Research Clinical Gastroenterology

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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 inflammation resulting from liver fluke infection, hepatitis and other inflammatory 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.: þ65 66 011324. E-mail addresses: [email protected], [email protected] (S. Kongpetch), [email protected] (A. Jusakul), [email protected] (C.K. Ong), [email protected] (W.K. Lim), [email protected] (S.G. Rozen), [email protected] (P. Tan), [email protected] (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 flukes 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 fluke infestation [4]. Cirrhosis, hepatitis B (HBV) and hepatitis C viral (HCV) infection among other risk factors identified 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 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 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 inflammation 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 identified 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 specific 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 classification 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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suggest that cholangiocytes alone may not be sufficient 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 sufficient 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 finally progression to metastatic ICC [17]. Molecular and cellular pathogenesis As alluded to above, chronic infection and inflammation in the bile ducts play an important role in cholangiocarcinogenesis. Inflammation causes the release of proinflammatory cytokines leading to induction of nitric oxide synthase (iNOS), a generator of nitric oxide (NO) in cholangiocytes. NO produced in infected and inflamed 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 inflammation triggered by repeated Ov infection was reported to mediate iNOS-dependent DNA damage in intrahepatic bile duct epithelium and inflammatory 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 profiling 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 identified 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 reflect 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, significant gains of 2p, 5p, 22q and significant 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 modification 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 profiling 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 profiling of miRNA in CCA cell lines (HUCCT1 and MEC) revealed biliary epithelial cell-specific 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 amplifications, 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 identified 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 fibroblast 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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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 identification of FGFR and ROS1 translocations may impact patient management, it is likely that further high-throughput genomic profiling 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 significant clinical implications from diagnosis to therapy. Mutations in well-known cancer drivers such as TP53 and KRAS have been identified 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 profiling of CCA, identifying novel mutated genes and providing new insights into the genetic basis of CCA tumorigenesis [61,66,67]. The first study, using whole-exome sequencing of 8 Ov-related CCA, identified 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 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 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 modifiers. 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)-specific 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 significantly 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 significant increase in proliferation whereas overexpression of wild-type ARID1A led to retarded cell proliferation [66]. In recent years, IDH mutations in cancer have attracted significant 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, inflammation, 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 specific 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, inflammation is considered to be one of the key contributing factors in CCA development. 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 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-specific activation of KRASG12D was sufficient for the development of invasive ICC. It promoted metastatic liver tumorigenesis 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 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 specific- inactivation of SMAD4 and PTEN Chronic CCl4 exposure in TP53-deficient mice Liver-specific activation of KRAS Liver-specific 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 profiling 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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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.

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

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Pathogenesis of cholangiocarcinoma: From genetics to signalling pathways.

Cholangiocarcinoma (CCA) is a malignant tumour of bile duct epithelial cells with dismal prognosis and rising incidence. Chronic inflammation resultin...
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