REVIEW URRENT C OPINION
Genetics of Opisthorchis viverrini-related cholangiocarcinoma Apinya Jusakul a,b, Sarinya Kongpetch a,b,c,d, and Bin Tean Teh a,b,e
Purpose of review We review the genetic, epigenetic and transcriptional landscape of liver fluke (Opisthorchis viverrini, Ov)related cholangiocarcinoma (CCA). Its distinct alterations, as compared with non-Ov-related CCA may help shed light on its underlying molecular mechanisms. Recent findings Recent whole-exome and targeted sequencing not only confirmed frequent mutations in known CCA-related genes including TP53 (44%), KRAS (16.7%) and SMAD4 (16.7%), but also revealed mutations in novel CCA-related genes associated with chromatin remodeling [BAP1 (2.8%), ARID1A (17.6%), MLL3 (13%) and IDH1/2 (2.8%)], WNT signaling [RNF43 (9.3%) and PEG3 (5.6%)] and KRAS/G protein signaling [GNAS (9.3%) and ROBO2 (9.3%)]. Interestingly, there is a significant difference in the frequency of mutated genes between Ov-related CCA and non-Ov-related CCA, such as p53 and IDH1/2, reflecting the impact of cause on pathogenesis. Altered DNA methylation and transcriptional profiles associated with xenobiotic metabolism and pro-inflammatory responses were also found in Ov-related CCA. Summary Liver fluke-induced chronic inflammation plays a crucial role in cholangiocarcinogenesis, resulting in distinct signatures of genetic, epigenetic and transcriptional alterations. These alterations, when contrasted with non-Ov-related CCA, indicate a unique pathogenic process in Ov-related CCA and may have potential clinical implications on diagnostics, therapeutics and prevention. Keywords cholangiocarcinoma, liver fluke, Opisthorchis viverrini, TP53
INTRODUCTION Cholangiocarcinoma (CCA) is a fatal cancer that develops along the biliary tract and carries a very poor prognosis. Accounting for 10–25% of all primary liver cancers worldwide, the highest incidence of this aggressive malignancy is in the northeast region of Thailand, neighboring Laos and Cambodia. In Thailand alone, an estimated 5000 cases of CCA are diagnosed annually. However, its incidence is increasing in western countries for unknown reasons [1]. Well established risk factors include primary sclerosing cholangitis, hepatolithiasis and choledochal cysts, whereas less-established potential risk factors include inflammatory bowel disease, hepatitis C virus, hepatitis B virus, cirrhosis, diabetes, obesity, alcohol consumption and smoking [2 ]. The common thread of all these factors appears to be chronic inflammation in the biliary tract epithelia. In this review, we focus on Opisthorchis viverrini (Ov)-related CCA. Ov is a foodborne trematode that encysts as a metacercaria
in the cyprinoid fish. Infection occurs when individuals ingest raw or uncooked fish infected with the metacercariae. Adult Ov inhabits the biliary tract and can reside within the human host for over 10 years [3 ]. The International Agency for Research on Cancer of the WHO lists Ov as a group 1 carcinogen [4]. Ov infection induces acute and chronic inflammation that leads to hepatobiliary abnormalities. &
a
Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, bProgram in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, cDepartment of Pharmacology, Faculty of Medicine, dLiver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand and e Cancer Science Institute of Singapore, National University of Singapore, Singapore
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Correspondence to Bin Tean Teh, MD, PhD, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. Tel: +65 6601 1366; e-mail: [email protected] Curr Opin Gastroenterol 2015, 31:258–263 DOI:10.1097/MOG.0000000000000162 Volume 31 Number 3 May 2015
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Genetics of Opisthorchis viverrini-related CCA Jusakul et al.
KEY POINTS Chronic inflammation in the biliary tract epithelia caused by Ov infection is a well known predisposing factor of CCA in northeast of Thailand and neighboring Laos and Cambodia. Chronic infection and inflammation contribute to genetic, epigenetic and gene expression alteration in Ov-associated CCA. Exome and capture sequencing showed that, apart from known CCA-related mutant genes including TP53 (44%), KRAS (16.7%) and SMAD4 (16.7%), there were novel mutated genes especially those involved in chromatin biology and dynamics for example, BAP1 (2.8%), ARID1A (17.6%), MLL3 (13%) and IDH1/2 (2.8%). Comparison studies showed that p53 and SMAD4 have significantly higher mutations, whereas BAP1 and IDH lower mutations, in Ov-related CCA than non-Ovrelated CCA. Altered methylation phenotypes and transcriptional profiles associated with xenobiotic metabolism and proinflammatory responses were found in Ov-related CCA.
Three main mechanisms are proposed to be involved, mechanical damage to the biliary epithelia caused by the feeding activities of the parasites, immunopathology because of infection-related inflammation and the toxic effects of parasite excretory/secretory molecules [1]. In response to Ov infection, inflammatory cells are activated by proinflammatory cytokines and nitric oxide generated by inducible nitric oxide synthase. Nitric oxide produced in infected and inflamed tissues has been postulated to contribute to cholangiocarcinogenesis by causing damage to DNA and proteins [5–7], resulting in mutagenic changes. It can also stimulate cyclooxygenase-2 expression, which can promote cholangiocyte growth via activation of growth factors such as epidermal growth factor receptor, mitogen-activated protein kinases and interleukin-6 [8]. Thus, chronic Ov infection and inflammation leads to accumulation of genetic, epigenetic and transcriptional alterations in CCA. We review these data and further compare with those of non Ov-related CCA with the goal of better understanding the underlying oncogenic mechanisms in CCA.
21q and loss of fragment at 1p, 9p, 17q and 22q was reported in four independent studies using microsatellite markers, each analyzing more than 50 cases of Ov-related CCA. Further analyses implicated the involvement of trefoil factor family 3 on 21q, run-related transcription factor 3 on 1p, CDKN2A on 9p, TP53 on 17p and thymidine phosphorylase on 22q [9–12]. Nevertheless, the pathogenicity of these findings has not been further corroborated by functional studies. In contrast, gain of chromosomal fragments 1q, 5q, 7p, 8q, 17q and 20q and loss of chromosomal fragments 1p, 3p, 4q, 6q, 8p, 9p, 17p and 18q were reported to be frequently found in 98 non-Ov-related CCA [13–15]. Comparison between the two suggests that gain of 22q and loss of 21p are preferentially observed in CCA associated with Ov, whereas loss of 1p, 9p and 17p are common aberrations shared between Ov-related CCA and non-Ov-related CCA, although a direct comparison of the two groups in the same technical platform would be required to confirm the distinction.
EPIGENETIC ALTERATION IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA Epigenetic alteration is one of the key molecular features of carcinogenesis, but in CCA, especially those that are Ov-related, limited studies have been carried out. A study using methylation-specific PCR revealed promoter hypermethylation of the mismatch repair gene hMLH1 in 44.6% of Ov-related CCA leading to its decreased expression and reduced function [16]. In another similar study, CpG-island hypermethylation of 26 loci was studied in 102 Ovrelated CCA and 29 matched adjacent normal tissues showing hypermethylation of 14–3–3s, OPCML, SFRP1, HIC1, PTEN and DcR1 (81.4, 72.5, 63.7, 38.2, 35.3 and 28.4%, respectively) [17]. The methylation of OPCML, a stress-responsive gene and p53-responsive gene, may serve as a methylation biomarker for CCA due to absence of methylation in normal adjacent tissue [18]. More recently, DNA methylation levels at a genome-wide scale were studied in 28 Ov-related CCA. The genes associated with a more stem cell-like phenotype were significantly hypermethylated at CpG sites compared with adjacent normal tissue. These genes include homeobox genes (HOXA9 and HOXD9) and target genes of polycomb repressive complex 2 [19 ]. Repression of these genes leads to loss of differentiation properties, supporting the hypothesis that cancer cells arise from undifferentiated progenitor cells or, alternatively, that cancer cells can undergo dedifferentiation during development. &
CHROMOSOMAL CHANGES To date, a comprehensive study of chromosomal and copy number variations in Ov-related CCA has not been conducted. A gain of fragment at
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MICRORNA PROFILING IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA MicroRNAs (miRs) are short, noncoding RNAs that function as posttranscriptional regulators by interacting with the 30 untranslated region of target genes and promoting translational repression or degradation of mRNAs. Aberrant miR expression has been identified in the progression of various cancers [20]. Recently, a comprehensive miR profiling was performed in 16 Ov-related intrahepatic CCA. Each histologic grade was found to be correlated with a distinct miR profile. Moderately differentiated intrahepatic Ov-related CCA showed the greatest miR deregulation in quantity and magnitude, followed by the papillary subtype, and finally well differentiated CCA. There were six miRs commonly deregulated in Ov-related CCA, including miR-135b, -141, -200c, -21, -221 and -222 [21 ]. Interestingly, deregulation of miR-21 was also described in non-Ov-related CCA [22]. The oncogenic function of miR-21 by inhibiting proapoptotic protein was reported in both Ov-related CCA and non-Ov-related CCA [23,24 ], suggesting that miR-21 is the common onco-miR in cholangiocarcinogenesis. &&
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GENETIC MUTATIONS IN OPISTHORCHIS VIVERRINI-ASSOCIATED CHOLANGIOCARCINOMA Recently, whole-exome sequencing of Thai Ovrelated CCA revealed 206 confirmed somatic mutations in 187 genes [25]. From the 15 genes selected for further analysis in additional cases, recurrently mutated genes in Ov-related CCA can be functionally grouped into seven biological processes or pathways, genome stability, transforming growth factor beta (TGF-b)/SMAD4 signaling, KRAS
Ov-related status
Ov-related CCA
TP53 SMAD4 KRAS ROBO2 GNAS ARID1A MLL3 BAP1 IDH1 IDH2 RNF43 PEG3 CDKN2A PTEN
and G protein signaling, epigenetic regulation, WNT signaling, cell cycle control and AKT and PI3K signaling (Fig. 1) [25,26 ]. Of note, mutations in well known cancer-related genes such as TP53 and KRAS were found in Ov-related CCA. TP53 mutations were the most frequent (44.4%). In TP53 mutant mice, addition of carbon tetrachloride caused the progression of epithelial hyperplasia of bile duct to malignant Intrahepatic CCA [27]. Activating KRAS (a GTPase belonging to the RAS superfamily) mutations were found in both intrahepatic CCA and extrahepatic CCA, ranging in frequency from 7 to 50% [25,28,29]. In addition, GNAS (a stimulatory G protein a subunit) mutations were found in Ov-related CCA (9.3%) (Table 1) [25,26 ,30 ]. Activating mutations in GNAS at codon 201 were identified in Ov-related cohort and have been reported in intraductal papillary mucinous neoplasms of the pancreas, especially in invasive lesions [31]. Taken together, genomic instability and RAS/RAF pathway alterations may play an important role in CCA tumorigenesis. Moreover, CDKN2A, a well known tumor suppressor gene, was mutated in 5.6% of Ov-related CCA. CDKN2A is a negative regulatory protein that controls the cell cycle. Homozygous deletion of the CDKN2A region was found in 5% of CCAs, with loss of heterozygosity present in 20% [32]. Besides these mutations, novel somatic mutations were also identified in the following genes: SMAD4 (16.7%), MLL3 (14.8%), ROBO2 (9.3%), RNF43 (9.3%), PEG3 (5.6%) and GNAS (9.3%) (Table 1). Interestingly, the frequency of SMAD4 mutations in Ov-related CCA is similar to that of KRAS. SMAD4 is a tumor suppressor gene that regulates cell cycle inhibition and apoptosis induction and is a crucial component in TGF-b signaling pathway [33]. Previously, inactivation of SMAD4 gene was found in 35% of intrahepatic CCA and 50% of extrahepatic CCA [34]. &&
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&&
Non Ov-related CCA
Mutated pathways Genomic stability TGF- β/SMAD4 signaling KRAS/G protein signaling
Epigenetic regulation
Wnt signaling Cell cycle control AKT/PI3K signaling
FIGURE 1. Somatic mutations and mutated pathways in Opisthorchis viverrini (Ov)-related and non-Ov-related cholangiocarcinoma (CCA). Left column indicates genes validated in Ong et al. [25] and Chan-On et al. [26 ] and top row indicates Ov-related status. Samples with or without mutations are labeled in black or white, respectively. &&
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Genetics of Opisthorchis viverrini-related CCA Jusakul et al. Table 1. Commonly mutated genes in Opisthorchis viverrini-related cholangiocarcinoma and non-Opisthorchis viverrinirelated cholangiocarcinoma Ov-related CCA
Non-Ov-related CCA
Thailand (N ¼ 54)a
Thailand (N ¼ 108)b
TP53
44.4
KRAS
16.7
SMAD4
Genes
CDKN2A MLL3
Singapore þ Romania (N ¼ 86)b
Europe (N ¼ 32)c
39.8
9.3
6
13.9
11.6
3
16.7
19.4
5.8
0
5.6
NR
NR
3
14.8
13.0
3.5
NR
ROBO2
9.3
5.6
2.3
NR
GNAS
9.3
5.6
0
NR
RNF43
9.3
7.4
3.5
NR
PTEN
3.7
NR
0
6
BAP1
NR
2.8
10.5
25
ARID1A
NR
17.6
10.5
19
IDH1/2
NR
2.8
9.3
19
PBRM1
NR
NR
NR
17
CCA, cholangiocarcinoma; NR, not reported. Data represented in table are in percentages. a Ong et al. [25]. && b Chan-On et al. [26 ]. && c Jiao et al. [30 ].
Several newly mutated genes are involved in WNT signaling pathway. RNF43 and PEG3 are negative regulators of WNT [35,36] with roles in regulating genomic stability though p53 regulation. RNF43 is a RING domain E3 ubiquitin ligase that interacts with NEDL1 and TP53 and suppresses p53-mediated apoptosis [37]. RNF43 mutations associated with poor survival were also observed in the Ov-related CCA. PEG3, on the other hand encodes product that induces apoptosis through interaction with Siah1a, an E3 ubiquitin ligase. Inhibition of PEG3 activity blocks p53-induced apoptosis [38]. Of note, 52% of the Ov-related CCA harbored mutations in at least one of these three genes TP53, RNF43 and PEG3 [25]. Another novel mutated gene is, ROBO2, which is a receptor protein involved in activating the Slit– Robo signaling pathway. Total or partial loss of the cytoplasmic domain of the ROBO2 protein leads to a failure to inhibit signaling for growth and proliferation. ROBO2 plays a role in axon guidance and modulates WNT signaling [39]. Notably, a novel class of mutated genes identified in Ov-related CCA includes those involved in chromatin biology and dynamics such as, MLL3, BAP1, ARID1A and IDH1/2. The frequency of somatic MLL3 mutations in Ov-related CCA is similar to that of SMAD4 and KRAS (Table 1), suggesting an important role for MLL3 in CCA. MLL3 is a histone modifier that encodes a histone-lysine N-methyltransferase. Truncating
mutations in MLL3 are predicted to result in protein products lacking the methyltransferase domain, suggestive of tumor suppressor gene [40]. Besides MLL3, a nucleosome remodeling complex known as the SWI/SNF complex appears to play a crucial role in cholangiocarcinogenesis. It mediates ATPdependent chromatin remodeling processes and exists in two forms, BAF (BRG1-associated or hbrm-associated factors) and PBAF (polybromoassociated BAF) [41]. Frequent ARID1A (a subunit of BAF complex) mutations were reported in both Ov-related (17.6%) and non-Ov-related (10.5–19%) CCA [25,26 ,30 ]. Knockdown of ARID1A in CCA cell lines resulted in a significant increase in proliferation whereas overexpression of wild-type ARID1A led to retarded cell proliferation. However, the mechanisms by which the mutations in these complexes drive tumorigenesis remain unclear. In addition, inactivating mutations of PBRM1, also a subunit of PBAF complex, were reported in non-Ovrelated CCA (17%) [30 ] but not in Ov-related CCA (Table 1). BAP1 and IDH1/2 mutations were also identified in CCA but their frequencies clearly varied according to underlying cause and geographical regions. BAP1 mutations (a member of the ubiquitin C-terminal hydrolases, subfamily of deubiquitylating enzymes) were found in 2.8% of Ov-related CCA, compared to 10.5–25% in non Ov-related CCA (Table 1). Increased cell proliferation was observed after BAP1 knockdown whereas overexpression of
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wild-type BAP1 in non-Ov-related CCA cell lines significantly suppressed cell proliferation [26 ]. IDH1/2 mutations were found in 2.8% of Ov-related CCA, compared with 10.5-19% in non-Ov-related CCAs (Table 1). A hypermethylation phenotype was clearly identified in IDH1/2-mutated CCA tumors [26 ,42 ], supporting the impact of IDH1/2 mutations on global DNA methylation. Once again to support their critical roles in CCA tumorigenesis, the majority of these mutations in MLL3, BAP1, ARID1A and IDH1/2 are found to be mutually exclusive to mutations of TP53, KRAS and SMAD4. &&
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TRANSCRIPTIONAL PROFILING IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA Several gene expression profiling studies have been conducted on Ov-related CCA. A recent study compared the gene expression profiles of 20 Thai Ovrelated CCA and 20 Japanese non-Ov-related CCA [43]. Genes involved in xenobiotic metabolism, such as the UDP glucuronosyltransferase 2 family: polypeptide B11 (UGT2B11), the UDP glucuronosyltransferase 1 family: polypeptide A10 (UGT1A10), carbohydrate (keratan sulfate Gal-6) sulfotransferase 4 (CHST4) and the sulfotransferase family: cytosolic, 1C, member 1 (SULT1C1) were upregulated in Ovrelated CCAs and thus were proposed as putative cause-specific markers in Ov-related CCA. Genotoxic stress induced by Ov infection may contribute to activation of the detoxification mechanisms. Moreover, increased expression of inflammatoryrelated genes such as interleukin 1 receptor was also reported in Ov-related CCA cell lines compared with non-Ov-related CCA cell lines, supporting the hypothesis that activation of inflammatory response is a factor of initiation and progression of Ov-related CCA [44 ]. In contrast, non-Ov-related CCA showed upregulation of genes related to growth factor signaling, such as TGF beta-induced protein, placental growth factor and insulin-like growth factor binding proteins 1 and 3. These findings demonstrate the presence of distinct patterns of underlying molecular pathogenesis in CCAs of different etiologies. &
CONCLUSION Ov infection is one of the main predisposing factors for cholangiocarcinogenesis. The pathogenesis of Ov infection mainly involves the induction of inflammation, which may result in DNA damage and genetic alterations leading to neoplastic transformation. Interestingly, there is a distinct pattern of gene mutations, chromosomal aberrations and epigenetic alterations in CCAs of different 262
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etiologies. Whole-exome sequencing studies show that commonly mutated genes in Ov-related CCA can be grouped into at least seven biological classes of differing functions, genome stability, TGF-b/ SMAD4 signaling, KRAS and G protein signaling, epigenetic regulation, WNT signaling, cell cycle control and protein kinase B (AKT) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Notably, TP53, KRAS and SMAD4 mutations are highly frequent in Ov-related CCA. Chromatin enzymes including MLL3, BAP1, ARID1A and IDH1/ 2 genes are found frequently mutated. Gene expression profiling showed the activation of xenobiotic metabolism and inflammatory response in Ovrelated CCA. Taken together, findings from comprehensive analyses of the genomic and transcriptional alterations in CCAs of different cause not only shed light on the underlying biology of these fatal malignancies, but also provide hints on potential avenues to improve prevention, diagnosis and targeted therapy. Acknowledgements The authors thank Dr Choon Kiat Ong, Dr Weng Khong Lim, Prof Patrick Tan and Assoc Prof Steven G. Rozen for their assistance with the study. Financial support and sponsorship This work was supported in part by funding from the Singapore National Medical Research Council (NMRC/ STAR/0006/2009), the Singapore Millennium Foundation, the Lee Foundation, the Tanoto Foundation, the Singapore National Cancer Centre Research Fund, the Duke-NUS Graduate Medical School, the Cancer Science Institute, Singapore and the Verdant Foundation, Hong Kong. Conflicts of interest There are no conflicts of interest.
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Genetics of Opisthorchis viverrini-related cholangiocarcinoma Apinya Jusakul a,b, Sarinya Kongpetch a,b,c,d, and Bin Tean Teh a,b,e
Purpose of review We review the genetic, epigenetic and transcriptional landscape of liver fluke (Opisthorchis viverrini, Ov)related cholangiocarcinoma (CCA). Its distinct alterations, as compared with non-Ov-related CCA may help shed light on its underlying molecular mechanisms. Recent findings Recent whole-exome and targeted sequencing not only confirmed frequent mutations in known CCA-related genes including TP53 (44%), KRAS (16.7%) and SMAD4 (16.7%), but also revealed mutations in novel CCA-related genes associated with chromatin remodeling [BAP1 (2.8%), ARID1A (17.6%), MLL3 (13%) and IDH1/2 (2.8%)], WNT signaling [RNF43 (9.3%) and PEG3 (5.6%)] and KRAS/G protein signaling [GNAS (9.3%) and ROBO2 (9.3%)]. Interestingly, there is a significant difference in the frequency of mutated genes between Ov-related CCA and non-Ov-related CCA, such as p53 and IDH1/2, reflecting the impact of cause on pathogenesis. Altered DNA methylation and transcriptional profiles associated with xenobiotic metabolism and pro-inflammatory responses were also found in Ov-related CCA. Summary Liver fluke-induced chronic inflammation plays a crucial role in cholangiocarcinogenesis, resulting in distinct signatures of genetic, epigenetic and transcriptional alterations. These alterations, when contrasted with non-Ov-related CCA, indicate a unique pathogenic process in Ov-related CCA and may have potential clinical implications on diagnostics, therapeutics and prevention. Keywords cholangiocarcinoma, liver fluke, Opisthorchis viverrini, TP53
INTRODUCTION Cholangiocarcinoma (CCA) is a fatal cancer that develops along the biliary tract and carries a very poor prognosis. Accounting for 10–25% of all primary liver cancers worldwide, the highest incidence of this aggressive malignancy is in the northeast region of Thailand, neighboring Laos and Cambodia. In Thailand alone, an estimated 5000 cases of CCA are diagnosed annually. However, its incidence is increasing in western countries for unknown reasons [1]. Well established risk factors include primary sclerosing cholangitis, hepatolithiasis and choledochal cysts, whereas less-established potential risk factors include inflammatory bowel disease, hepatitis C virus, hepatitis B virus, cirrhosis, diabetes, obesity, alcohol consumption and smoking [2 ]. The common thread of all these factors appears to be chronic inflammation in the biliary tract epithelia. In this review, we focus on Opisthorchis viverrini (Ov)-related CCA. Ov is a foodborne trematode that encysts as a metacercaria
in the cyprinoid fish. Infection occurs when individuals ingest raw or uncooked fish infected with the metacercariae. Adult Ov inhabits the biliary tract and can reside within the human host for over 10 years [3 ]. The International Agency for Research on Cancer of the WHO lists Ov as a group 1 carcinogen [4]. Ov infection induces acute and chronic inflammation that leads to hepatobiliary abnormalities. &
a
Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, bProgram in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, cDepartment of Pharmacology, Faculty of Medicine, dLiver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, Thailand and e Cancer Science Institute of Singapore, National University of Singapore, Singapore
&
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Correspondence to Bin Tean Teh, MD, PhD, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore. Tel: +65 6601 1366; e-mail: [email protected] Curr Opin Gastroenterol 2015, 31:258–263 DOI:10.1097/MOG.0000000000000162 Volume 31 Number 3 May 2015
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Genetics of Opisthorchis viverrini-related CCA Jusakul et al.
KEY POINTS Chronic inflammation in the biliary tract epithelia caused by Ov infection is a well known predisposing factor of CCA in northeast of Thailand and neighboring Laos and Cambodia. Chronic infection and inflammation contribute to genetic, epigenetic and gene expression alteration in Ov-associated CCA. Exome and capture sequencing showed that, apart from known CCA-related mutant genes including TP53 (44%), KRAS (16.7%) and SMAD4 (16.7%), there were novel mutated genes especially those involved in chromatin biology and dynamics for example, BAP1 (2.8%), ARID1A (17.6%), MLL3 (13%) and IDH1/2 (2.8%). Comparison studies showed that p53 and SMAD4 have significantly higher mutations, whereas BAP1 and IDH lower mutations, in Ov-related CCA than non-Ovrelated CCA. Altered methylation phenotypes and transcriptional profiles associated with xenobiotic metabolism and proinflammatory responses were found in Ov-related CCA.
Three main mechanisms are proposed to be involved, mechanical damage to the biliary epithelia caused by the feeding activities of the parasites, immunopathology because of infection-related inflammation and the toxic effects of parasite excretory/secretory molecules [1]. In response to Ov infection, inflammatory cells are activated by proinflammatory cytokines and nitric oxide generated by inducible nitric oxide synthase. Nitric oxide produced in infected and inflamed tissues has been postulated to contribute to cholangiocarcinogenesis by causing damage to DNA and proteins [5–7], resulting in mutagenic changes. It can also stimulate cyclooxygenase-2 expression, which can promote cholangiocyte growth via activation of growth factors such as epidermal growth factor receptor, mitogen-activated protein kinases and interleukin-6 [8]. Thus, chronic Ov infection and inflammation leads to accumulation of genetic, epigenetic and transcriptional alterations in CCA. We review these data and further compare with those of non Ov-related CCA with the goal of better understanding the underlying oncogenic mechanisms in CCA.
21q and loss of fragment at 1p, 9p, 17q and 22q was reported in four independent studies using microsatellite markers, each analyzing more than 50 cases of Ov-related CCA. Further analyses implicated the involvement of trefoil factor family 3 on 21q, run-related transcription factor 3 on 1p, CDKN2A on 9p, TP53 on 17p and thymidine phosphorylase on 22q [9–12]. Nevertheless, the pathogenicity of these findings has not been further corroborated by functional studies. In contrast, gain of chromosomal fragments 1q, 5q, 7p, 8q, 17q and 20q and loss of chromosomal fragments 1p, 3p, 4q, 6q, 8p, 9p, 17p and 18q were reported to be frequently found in 98 non-Ov-related CCA [13–15]. Comparison between the two suggests that gain of 22q and loss of 21p are preferentially observed in CCA associated with Ov, whereas loss of 1p, 9p and 17p are common aberrations shared between Ov-related CCA and non-Ov-related CCA, although a direct comparison of the two groups in the same technical platform would be required to confirm the distinction.
EPIGENETIC ALTERATION IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA Epigenetic alteration is one of the key molecular features of carcinogenesis, but in CCA, especially those that are Ov-related, limited studies have been carried out. A study using methylation-specific PCR revealed promoter hypermethylation of the mismatch repair gene hMLH1 in 44.6% of Ov-related CCA leading to its decreased expression and reduced function [16]. In another similar study, CpG-island hypermethylation of 26 loci was studied in 102 Ovrelated CCA and 29 matched adjacent normal tissues showing hypermethylation of 14–3–3s, OPCML, SFRP1, HIC1, PTEN and DcR1 (81.4, 72.5, 63.7, 38.2, 35.3 and 28.4%, respectively) [17]. The methylation of OPCML, a stress-responsive gene and p53-responsive gene, may serve as a methylation biomarker for CCA due to absence of methylation in normal adjacent tissue [18]. More recently, DNA methylation levels at a genome-wide scale were studied in 28 Ov-related CCA. The genes associated with a more stem cell-like phenotype were significantly hypermethylated at CpG sites compared with adjacent normal tissue. These genes include homeobox genes (HOXA9 and HOXD9) and target genes of polycomb repressive complex 2 [19 ]. Repression of these genes leads to loss of differentiation properties, supporting the hypothesis that cancer cells arise from undifferentiated progenitor cells or, alternatively, that cancer cells can undergo dedifferentiation during development. &
CHROMOSOMAL CHANGES To date, a comprehensive study of chromosomal and copy number variations in Ov-related CCA has not been conducted. A gain of fragment at
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Biliary tract
MICRORNA PROFILING IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA MicroRNAs (miRs) are short, noncoding RNAs that function as posttranscriptional regulators by interacting with the 30 untranslated region of target genes and promoting translational repression or degradation of mRNAs. Aberrant miR expression has been identified in the progression of various cancers [20]. Recently, a comprehensive miR profiling was performed in 16 Ov-related intrahepatic CCA. Each histologic grade was found to be correlated with a distinct miR profile. Moderately differentiated intrahepatic Ov-related CCA showed the greatest miR deregulation in quantity and magnitude, followed by the papillary subtype, and finally well differentiated CCA. There were six miRs commonly deregulated in Ov-related CCA, including miR-135b, -141, -200c, -21, -221 and -222 [21 ]. Interestingly, deregulation of miR-21 was also described in non-Ov-related CCA [22]. The oncogenic function of miR-21 by inhibiting proapoptotic protein was reported in both Ov-related CCA and non-Ov-related CCA [23,24 ], suggesting that miR-21 is the common onco-miR in cholangiocarcinogenesis. &&
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GENETIC MUTATIONS IN OPISTHORCHIS VIVERRINI-ASSOCIATED CHOLANGIOCARCINOMA Recently, whole-exome sequencing of Thai Ovrelated CCA revealed 206 confirmed somatic mutations in 187 genes [25]. From the 15 genes selected for further analysis in additional cases, recurrently mutated genes in Ov-related CCA can be functionally grouped into seven biological processes or pathways, genome stability, transforming growth factor beta (TGF-b)/SMAD4 signaling, KRAS
Ov-related status
Ov-related CCA
TP53 SMAD4 KRAS ROBO2 GNAS ARID1A MLL3 BAP1 IDH1 IDH2 RNF43 PEG3 CDKN2A PTEN
and G protein signaling, epigenetic regulation, WNT signaling, cell cycle control and AKT and PI3K signaling (Fig. 1) [25,26 ]. Of note, mutations in well known cancer-related genes such as TP53 and KRAS were found in Ov-related CCA. TP53 mutations were the most frequent (44.4%). In TP53 mutant mice, addition of carbon tetrachloride caused the progression of epithelial hyperplasia of bile duct to malignant Intrahepatic CCA [27]. Activating KRAS (a GTPase belonging to the RAS superfamily) mutations were found in both intrahepatic CCA and extrahepatic CCA, ranging in frequency from 7 to 50% [25,28,29]. In addition, GNAS (a stimulatory G protein a subunit) mutations were found in Ov-related CCA (9.3%) (Table 1) [25,26 ,30 ]. Activating mutations in GNAS at codon 201 were identified in Ov-related cohort and have been reported in intraductal papillary mucinous neoplasms of the pancreas, especially in invasive lesions [31]. Taken together, genomic instability and RAS/RAF pathway alterations may play an important role in CCA tumorigenesis. Moreover, CDKN2A, a well known tumor suppressor gene, was mutated in 5.6% of Ov-related CCA. CDKN2A is a negative regulatory protein that controls the cell cycle. Homozygous deletion of the CDKN2A region was found in 5% of CCAs, with loss of heterozygosity present in 20% [32]. Besides these mutations, novel somatic mutations were also identified in the following genes: SMAD4 (16.7%), MLL3 (14.8%), ROBO2 (9.3%), RNF43 (9.3%), PEG3 (5.6%) and GNAS (9.3%) (Table 1). Interestingly, the frequency of SMAD4 mutations in Ov-related CCA is similar to that of KRAS. SMAD4 is a tumor suppressor gene that regulates cell cycle inhibition and apoptosis induction and is a crucial component in TGF-b signaling pathway [33]. Previously, inactivation of SMAD4 gene was found in 35% of intrahepatic CCA and 50% of extrahepatic CCA [34]. &&
&&
&&
Non Ov-related CCA
Mutated pathways Genomic stability TGF- β/SMAD4 signaling KRAS/G protein signaling
Epigenetic regulation
Wnt signaling Cell cycle control AKT/PI3K signaling
FIGURE 1. Somatic mutations and mutated pathways in Opisthorchis viverrini (Ov)-related and non-Ov-related cholangiocarcinoma (CCA). Left column indicates genes validated in Ong et al. [25] and Chan-On et al. [26 ] and top row indicates Ov-related status. Samples with or without mutations are labeled in black or white, respectively. &&
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Genetics of Opisthorchis viverrini-related CCA Jusakul et al. Table 1. Commonly mutated genes in Opisthorchis viverrini-related cholangiocarcinoma and non-Opisthorchis viverrinirelated cholangiocarcinoma Ov-related CCA
Non-Ov-related CCA
Thailand (N ¼ 54)a
Thailand (N ¼ 108)b
TP53
44.4
KRAS
16.7
SMAD4
Genes
CDKN2A MLL3
Singapore þ Romania (N ¼ 86)b
Europe (N ¼ 32)c
39.8
9.3
6
13.9
11.6
3
16.7
19.4
5.8
0
5.6
NR
NR
3
14.8
13.0
3.5
NR
ROBO2
9.3
5.6
2.3
NR
GNAS
9.3
5.6
0
NR
RNF43
9.3
7.4
3.5
NR
PTEN
3.7
NR
0
6
BAP1
NR
2.8
10.5
25
ARID1A
NR
17.6
10.5
19
IDH1/2
NR
2.8
9.3
19
PBRM1
NR
NR
NR
17
CCA, cholangiocarcinoma; NR, not reported. Data represented in table are in percentages. a Ong et al. [25]. && b Chan-On et al. [26 ]. && c Jiao et al. [30 ].
Several newly mutated genes are involved in WNT signaling pathway. RNF43 and PEG3 are negative regulators of WNT [35,36] with roles in regulating genomic stability though p53 regulation. RNF43 is a RING domain E3 ubiquitin ligase that interacts with NEDL1 and TP53 and suppresses p53-mediated apoptosis [37]. RNF43 mutations associated with poor survival were also observed in the Ov-related CCA. PEG3, on the other hand encodes product that induces apoptosis through interaction with Siah1a, an E3 ubiquitin ligase. Inhibition of PEG3 activity blocks p53-induced apoptosis [38]. Of note, 52% of the Ov-related CCA harbored mutations in at least one of these three genes TP53, RNF43 and PEG3 [25]. Another novel mutated gene is, ROBO2, which is a receptor protein involved in activating the Slit– Robo signaling pathway. Total or partial loss of the cytoplasmic domain of the ROBO2 protein leads to a failure to inhibit signaling for growth and proliferation. ROBO2 plays a role in axon guidance and modulates WNT signaling [39]. Notably, a novel class of mutated genes identified in Ov-related CCA includes those involved in chromatin biology and dynamics such as, MLL3, BAP1, ARID1A and IDH1/2. The frequency of somatic MLL3 mutations in Ov-related CCA is similar to that of SMAD4 and KRAS (Table 1), suggesting an important role for MLL3 in CCA. MLL3 is a histone modifier that encodes a histone-lysine N-methyltransferase. Truncating
mutations in MLL3 are predicted to result in protein products lacking the methyltransferase domain, suggestive of tumor suppressor gene [40]. Besides MLL3, a nucleosome remodeling complex known as the SWI/SNF complex appears to play a crucial role in cholangiocarcinogenesis. It mediates ATPdependent chromatin remodeling processes and exists in two forms, BAF (BRG1-associated or hbrm-associated factors) and PBAF (polybromoassociated BAF) [41]. Frequent ARID1A (a subunit of BAF complex) mutations were reported in both Ov-related (17.6%) and non-Ov-related (10.5–19%) CCA [25,26 ,30 ]. Knockdown of ARID1A in CCA cell lines resulted in a significant increase in proliferation whereas overexpression of wild-type ARID1A led to retarded cell proliferation. However, the mechanisms by which the mutations in these complexes drive tumorigenesis remain unclear. In addition, inactivating mutations of PBRM1, also a subunit of PBAF complex, were reported in non-Ovrelated CCA (17%) [30 ] but not in Ov-related CCA (Table 1). BAP1 and IDH1/2 mutations were also identified in CCA but their frequencies clearly varied according to underlying cause and geographical regions. BAP1 mutations (a member of the ubiquitin C-terminal hydrolases, subfamily of deubiquitylating enzymes) were found in 2.8% of Ov-related CCA, compared to 10.5–25% in non Ov-related CCA (Table 1). Increased cell proliferation was observed after BAP1 knockdown whereas overexpression of
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wild-type BAP1 in non-Ov-related CCA cell lines significantly suppressed cell proliferation [26 ]. IDH1/2 mutations were found in 2.8% of Ov-related CCA, compared with 10.5-19% in non-Ov-related CCAs (Table 1). A hypermethylation phenotype was clearly identified in IDH1/2-mutated CCA tumors [26 ,42 ], supporting the impact of IDH1/2 mutations on global DNA methylation. Once again to support their critical roles in CCA tumorigenesis, the majority of these mutations in MLL3, BAP1, ARID1A and IDH1/2 are found to be mutually exclusive to mutations of TP53, KRAS and SMAD4. &&
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TRANSCRIPTIONAL PROFILING IN OPISTHORCHIS VIVERRINI-RELATED CHOLANGIOCARCINOMA Several gene expression profiling studies have been conducted on Ov-related CCA. A recent study compared the gene expression profiles of 20 Thai Ovrelated CCA and 20 Japanese non-Ov-related CCA [43]. Genes involved in xenobiotic metabolism, such as the UDP glucuronosyltransferase 2 family: polypeptide B11 (UGT2B11), the UDP glucuronosyltransferase 1 family: polypeptide A10 (UGT1A10), carbohydrate (keratan sulfate Gal-6) sulfotransferase 4 (CHST4) and the sulfotransferase family: cytosolic, 1C, member 1 (SULT1C1) were upregulated in Ovrelated CCAs and thus were proposed as putative cause-specific markers in Ov-related CCA. Genotoxic stress induced by Ov infection may contribute to activation of the detoxification mechanisms. Moreover, increased expression of inflammatoryrelated genes such as interleukin 1 receptor was also reported in Ov-related CCA cell lines compared with non-Ov-related CCA cell lines, supporting the hypothesis that activation of inflammatory response is a factor of initiation and progression of Ov-related CCA [44 ]. In contrast, non-Ov-related CCA showed upregulation of genes related to growth factor signaling, such as TGF beta-induced protein, placental growth factor and insulin-like growth factor binding proteins 1 and 3. These findings demonstrate the presence of distinct patterns of underlying molecular pathogenesis in CCAs of different etiologies. &
CONCLUSION Ov infection is one of the main predisposing factors for cholangiocarcinogenesis. The pathogenesis of Ov infection mainly involves the induction of inflammation, which may result in DNA damage and genetic alterations leading to neoplastic transformation. Interestingly, there is a distinct pattern of gene mutations, chromosomal aberrations and epigenetic alterations in CCAs of different 262
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etiologies. Whole-exome sequencing studies show that commonly mutated genes in Ov-related CCA can be grouped into at least seven biological classes of differing functions, genome stability, TGF-b/ SMAD4 signaling, KRAS and G protein signaling, epigenetic regulation, WNT signaling, cell cycle control and protein kinase B (AKT) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Notably, TP53, KRAS and SMAD4 mutations are highly frequent in Ov-related CCA. Chromatin enzymes including MLL3, BAP1, ARID1A and IDH1/ 2 genes are found frequently mutated. Gene expression profiling showed the activation of xenobiotic metabolism and inflammatory response in Ovrelated CCA. Taken together, findings from comprehensive analyses of the genomic and transcriptional alterations in CCAs of different cause not only shed light on the underlying biology of these fatal malignancies, but also provide hints on potential avenues to improve prevention, diagnosis and targeted therapy. Acknowledgements The authors thank Dr Choon Kiat Ong, Dr Weng Khong Lim, Prof Patrick Tan and Assoc Prof Steven G. Rozen for their assistance with the study. Financial support and sponsorship This work was supported in part by funding from the Singapore National Medical Research Council (NMRC/ STAR/0006/2009), the Singapore Millennium Foundation, the Lee Foundation, the Tanoto Foundation, the Singapore National Cancer Centre Research Fund, the Duke-NUS Graduate Medical School, the Cancer Science Institute, Singapore and the Verdant Foundation, Hong Kong. Conflicts of interest There are no conflicts of interest.
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