Malignant Cholangiocytes Research Articles

Serum ITIH5 as a novel diagnostic biomarker in cholangiocarcinoma.

Cholangiocarcinoma often remains undetected until advanced stages due to the lack of reliable diagnostic markers. Our goal was to identify a unique secretory protein for cholangiocarcinoma diagnosis and differentiation from other malignancies, benign hepatobiliary diseases, and chronic liver conditions. We conducted bulk RNA-seq analysis to identify genes specifically upregulated in cholangiocarcinoma but not in most other cancers, benign hepatobiliary diseases, and chronic liver diseases focusing on exocrine protein-encoding genes. Single-cell RNA sequencing examined subcellular distribution. Immunohistochemistry and enzyme-linked immunosorbent assays assessed tissue and serum expression. Diagnostic performance was evaluated via receiver-operating characteristic (ROC) analysis. Inter-alpha-trypsin inhibitor heavy chain family member five (ITIH5), a gene encoding an extracellular protein, is notably upregulated in cholangiocarcinoma. This elevation is not observed in most other cancer types, benign hepatobiliary diseases, or chronic liver disorders. It is specifically expressed by malignant cholangiocytes. ITIH5 expression in cholangiocarcinoma tissues exceeded that in nontumorous bile duct, hepatocellular carcinoma, and nontumorous hepatic tissues. Serum ITIH5 levels were elevated in cholangiocarcinoma compared with controls (hepatocellular carcinoma, benign diseases, chronic hepatitis B, and healthy individuals). ITIH5 yielded areas under the ROC curve (AUCs) from 0.839 to 0.851 distinguishing cholangiocarcinoma from controls. Combining ITIH5 with carbohydrate antigen 19-9 (CA19-9) enhanced CA19-9's diagnostic effectiveness. In conclusion, serum ITIH5 may serve as a novel noninvasive cholangiocarcinoma diagnostic marker.

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma

Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). EVs from patients with isolated PSC (n= 45), concomitant PSC-CCA (n= 44), PSC who developed CCA during follow-up (PSC to CCA; n= 25), CCAs from non-PSC aetiology (n= 56), and hepatocellular carcinoma (n= 34) and healthy individuals (n= 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC= 0.947; odds ratio [OR]=36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC= 0.992; OR= 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC= 0.941; OR= 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.

From the Editor’s Desk...

From the Editor’s Desk...

Different iron-handling in inflamed small and large cholangiocytes and in small and large-duct type intrahepatic cholangiocarcinoma.

Cholangiocarcinoma (CCA) represents the second most common primary hepatic malignancy and originates from the neoplastic transformation of the biliary cells. The intrahepatic subtype includes two morpho-molecular forms: large-duct type intrahepatic CCA (iCCA) and small-duct type iCCA. Iron is fundamental for the cellular processes, contributing in tumor development and progression. The aim of this study was to evaluate iron uptake, storage, and efflux proteins in both lipopolysaccharide-inflamed small and large cholangiocytes as well as in different iCCA subtypes. Our results show that, despite an increase in interleukin-6 production by both small and large cholangiocytes, ferroportin (Fpn) was decreased only in small cholangiocytes, whereas transferrin receptor-1 (TfR1) and ferritin (Ftn) did not show any change. Differently from in vitro models, Fpn expression was increased in malignant cholangiocytes of small-duct type iCCA in comparison to large-duct type iCCA and peritumoral tissues. TfR1, Ftn and hepcidin were enhanced, even if at different extent, in both malignant cholangiocytes in comparison to the surrounding samples. Lactoferrin was higher in large-duct type iCCA in respect to small-duct type iCCA and peritumoral tissues. These findings show a different iron handling by inflamed small and large cholangiocytes, and small and large-duct type iCCA. The difference in iron homeostasis by the iCCA subtypes may have implications for the tumor management.

Senescent cholangiocytes release extracellular vesicles that alter target cell phenotype via the epidermal growth factor receptor.

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells. EVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild-type and Mdr2-/- mice (a murine model of PSC), and assessed mouse monocyte activation. EVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2-/- mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF-dependent Interleukin 1-beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2-/- mouse plasma EVs induced activation of mouse monocytes. The data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.

Epidermal growth factor-like domain multiple 7 (EGFL7): Expression and possible effect on biliary epithelium growth in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. The intrahepatic subtype comprises two forms: mucin-iCCA and mixed-iCCA. Epidermal growth factor-like domain multiple (EGFL7) is overexpressed in less differentiated liver tumors. The aim of this study was to assess the presence of EGFL7 due to its possible role in the growth of CCA. Hematoxylin & Eosin and periodic acid- Schiff staining were used to evaluate the morphological aspects and glycogen deposition. Immunohistochemistry and immunofluorescence were performed to identify the presence of EGFL7 both in tumor sections ex vivo and in appropriate cell lines in culture. We found that EGFL7 is expressed in malignant cholangiocytes of mixed-iCCA and absent in mucin-iCCA. In conclusion the expression of EGFL7 might be useful in the classification of CCA subtypes.

Global identification and characterization of lncRNAs that control inflammation in malignant cholangiocytes

BackgroundLong noncoding RNAs (lncRNAs) are known to play important roles in different cell contexts, including cancers. However, little is known about lncRNAs in cholangiocarcinoma (CCA), a cholangiocyte malignancy with poor prognosis, and associated with chronic inflammation and damage to the biliary epithelium. This study determined whether lncRNAs were dysregulated and participated in disease diagnosis or pivotal inflammation pathways through a genome-wide lncRNA screening and functional analysis.ResultsWe firstly identified a large number of lncRNAs abnormally expressed between 9 pairs of cancerous and adjacent tissues of CCA, and between intra-hepatic CCA and extra-hepatic CCA through a genome-wide profiling. A set of aberrant differentially expressed lncRNAs were further validated in a training set (16 pairs) and a test set (11 pairs) of CCA patient samples. Following assessment of the diagnostic value of the 7 differentially expressed lncRNAs, we confirmed the optimal combination of H19, C3P1, AC005550.3, PVT1, and LPAL2 with area under the curve of 0.8828 [95% CI: 0.7441–1.021, P < 0.001], with 93.75% sensitivity and 81.25% specificity, at the cutoff point of − 0.2884 to distinguish the CCA tissue from the normal ones, suggesting that specific lncRNAs may have potential for detecting CCA. More importantly, the genome-wide locus and lncRNA/mRNA co-expression analyses revealed a set of lncRNAs that participated in inflammation and oxidative stress response pathways by regulating genes in cis or in trans. Finally, APOC1P1, PVT1, and LPAL2 were validated to regulate the migration and some pivotal inflammation genes under the CCA pathogenesis.ConclusionsOur findings are the first to show that lncRNAs may not only be potential biomarkers of CCA progression but also respond to inflammation in CCA.

Notch2 controls hepatocyte-derived cholangiocarcinoma formation in mice

Liver cancer comprises a group of malignant tumors, among which hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common. ICC is especially pernicious and associated with poor clinical outcome. Studies have shown that a subset of human ICCs may originate from mature hepatocytes. However, the mechanisms driving the trans-differentiation of hepatocytes into malignant cholangiocytes remain poorly defined. We adopted lineage tracing techniques and an established murine hepatocyte-derived ICC model by hydrodynamic injection of activated forms of AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes. Wild-type, Notch1flox/flox, and Notch2flox/flox mice were used to investigate the role of canonical Notch signaling and Notch receptors in AKT/Yap-driven ICC formation. Human ICC and HCC cell lines were transfected with siRNA against Notch2 to determine whether Notch2 regulates biliary marker expression in liver tumor cells. We found that AKT/Yap-induced ICC formation is hepatocyte derived and this process is strictly dependent on the canonical Notch signaling pathway in vivo. Deletion of Notch2 in AKT/Yap-induced tumors switched the phenotype from ICC to hepatocellular adenoma-like lesions, while inactivation of Notch1 in hepatocytes did not result in significant histomorphological changes. Finally, in vitro studies revealed that Notch2 silencing in ICC and HCC cell lines down-regulates the expression of Sox9 and EpCAM biliary markers. Notch2 is the major determinant of hepatocyte-derived ICC formation in mice.

Double immunostaining for maspin and p53 on cell blocks increases the diagnostic value of biliary brushing cytology.

Our objective is to elucidate the usefulness of maspin/p53 double immunostaining on biliary brushing cytology specimens. We first examined the expression of maspin in the biliary epithelium with variable degrees of dysplasia using surgically resected specimens (n = 56). Maspin appeared to be overexpressed in a stepwise manner from benign to malignant cholangiocytes: the reactive epithelium (20%), biliary intraepithelial neoplasia (~50%), and invasive cholangiocarcinomas (>90%). Next, an automated sequential double immunostaining protocol for maspin and p53 was applied to paraffin-embedded cell blocks of the biliary brushing cytology specimens obtained from 58 consecutive patients. Cell block preparation was successful in 44 cases (76%), which were morphologically diagnosed as adenocarcinoma (n = 16), atypical cells not diagnostic for malignancy (n = 10), and benign (n = 18). Double positive cells were observed in 14/16 (88%) morphologically malignant, 6/10 (60%) borderline, and 0/18 benign cases. All 20 positive cases were proven to have pancreatobiliary malignancies by subsequent imaging or pathological analyses. A similar staining protocol for S100P and p53 was also applied to the same cohort; however, the positive frequency was slightly lower than those of maspin and p53 (36% vs. 45%). In conclusion, Maspin/p53 double immunostaining on cell blocks contributes to the detection of malignant cells in biliary brushing cytology specimens.

Abstract 192: Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from the epithelial cells lining the biliary tract in the liver, the cholangiocytes. While cholangiocytes normally express cilia, we showed that they are significantly reduced in CCA by a mechanism involving overexpression of histone deacetylase 6 (HDAC6), a molecule that deacetylates the ciliary axoneme and promotes ciliary disassembly. Even though HDAC6 protein is overexpressed in CCA, we found no differences in its messenger RNA (mRNA) level, suggesting a post-transcriptional regulation, possibly involving microRNA (miRNA). Therefore, we hypothesized that decreased levels of specific miRNAs induce overexpression of HDAC6 in CCA. We compared miRNA levels between normal and CCA cholangiocyte cell lines and found that three miRNAs potentially targeting HDAC6 are downregulated (40-55% decrease) in CCA: miR-433, miR-22, and miR-200a. Experimental overexpression of miR-433, miR-22, or miR-200a in normal cholangiocytes decreased HDAC6 expression by 82%, 33%, and 39%, respectively. We selected miR-433 and miR-22 for further analysis and the downregulation of both miRNAs were confirmed by qRT-PCR on normal and six different CCA cell lines, and by in situ hybridization on control and CCA human liver samples. To further investigate the dysregulation of these miRNAs, we measured its precursor forms by qRT-PCR and, in contrast to the mature miR-433 and miR-22, the levels of the precursors were increased in malignant cells. In situ hybridization on human samples showed that the miR-433 probe was accumulated in the nucleus of malignant cholangiocytes. Taken together, these data suggested a potential impairment of nuclear export; therefore, we assessed the expression of the protein responsible for transporting microRNA precursors out of the nucleus, exportin-5. By western blotting, we found that exportin-5 is decreased by 50% in five different CCA cell lines compared to normal cholangiocyte cell lines. We confirmed the downregulation of exportin-5 protein by immunofluorescence of normal and CCA human liver samples. Finally, the experimental overexpression of exportin-5 in CCA cell lines restores the precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6, and decreased proliferation and anchorage independent growth of the malignant cells. In summary, these data suggest that downregulation of exportin-5, preventing the nuclear export of the precursor forms of miR-433 and miR-22, may be the underlying mechanism explaining the decrease of miR-433 and miR-22, and the overexpression of HDAC6 in CCA. Thus, our work further clarifies the molecular mechanisms accounting for the absence of cilia in CCA and also identifies multiple potential molecules for targeted therapies like HDAC6, miR-433, miR-22, or exportin-5. Citation Format: Maria J. Lorenzo Pisarello, Brynn N. Howard, Christy E. Trussoni, Sergio Gradilone. Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2015-192

Lactoferrin regulate biliary epithelium growth and the activation of hepatic progenitor cell niche

Lactoferin (Lf) is an iron-binding glycoprotein belonging to the transferrin family and it is present at high levels in breast milk and colostrum. This protein has many known functions and it is a potential antibacterial, antiviral, immunostimulatory, antioxidant, and cancer preventive agent.It has been seen that a 105 kDa Lf receptor (LfR) specifically mediates the effects of Lf in several different cell types (1). In human cholangiopathies, cholangiocytes are able to proliferate and replace the cell loss restoring the integrity of damaged biliary epithelium. However, when cholangiocyte proliferation is severely impaired, the activation of facultative hepatic progenitor cells (HPCs) takes place (2). The aims of our study have been i) to investigate the expression of Lf and LfR in cholangiocytes and in HPCs both in rats and in humans; and ii) to evaluate the in vitro effects of bLf on cholangiocyte proliferation and on HPC activation. Liver specimens have been obtained from normal (N=5) and bile duct ligated (BDL) (N=5) rats; from normal patients (N=5) and from patients with primary biliary cirrhosis (PBC, N=5); Specimens were processed for histology, immunohistochemistry and immunofluorescence. Moreover for the in vitro study small and large cholangioytes from mouse, human non malignant cholangiocytes (H69), and HPCs treated or not with lactoferrin were used. Our results showed that: i) cholangiocytes and hepatic progenitor cells express lactoferrin and its receptor, ii) cholangiocytes and HPC proliferation is enhanced by lactoferrin; iii) the treatment with lactoferrin determine the commitment of HPCs towards cholangiocyte fate; this commitment is characterized by HPC morphological and phenotypical changes. Our current findings suggest that modulation of lactoferrin may be an important therapeutic tool for managing the proliferation of cholangiocyte and the activation of progenitor cell compartment in biliary disorders.

Transcribed ultraconserved regions are aberrantly expressed and can be modulated by interleukin 6 in cholangiocarcinoma

BackgroundThe inflammation-associated cytokine interleukin (IL) 6 has been implicated in cholangiocarcinogenesis. Transcripts encoded by a group of highly conserved regions (transcribed-ultraconserved regions [T-UCRs]) are novel promising non-coding RNAs that proved to have a role in human carcinogenesis and liver cancer progression. We aimed to study expression of T-UCRs in cholangiocarcinoma. MethodsMzChA-1, TFK, CC-LP-1, and KMCH human cholangiocarcinoma cells, and normal human intrahepatic biliary epithelial cells (HIBEC) were used. T-UCR expression profiling was done with real-time PCR. IL-6 dependent T-UCR expression was assessed in Mz-ChA-1 cells incubated with 10 ng/L IL6 for 24 h or stably transfected with full-length IL6 (Mz-IL6). FindingsThe expression of 65 T-UCRs was altered in Mz-ChA-1 compared with normal HIBEC cells. Of these, 12 T-UCRs were upregulated by more than two fold and 48 T-UCRs were downregulated including ultraconserved RNA uc.169, which was dramatically reduced in other human cholangiocarcinoma cell lines and tissues. uc.169 was further reduced in Mz-ChA-1 cells overexpressing IL6. Incubation of MzChA-1 cells with IL6 reduced expression of uc.169, and pretreatment with the inhibitor of transcription actinomycin D inhibited this effect. Enforced expression of IL6 caused an increase in the microRNAs miR-21 and miR-26-a1, which are involved in promotion of cholangiocarcinoma. The uc.169 sequence includes target seed regions for miR-21 and miR-26-a1 and they may be responsible for the downregulation of uc.169. Overexpression of uc.169 in Mz-ChA-1 cells reduced cell proliferation and caused arrest in phase G0/1 of the cell cycle, with increased apoptosis. InterpretationWe report that T-UCRs are differentially expressed in malignant human cholangiocytes, and that IL6 can modulate the expression of selected T-UCRs, probably through a miRNA-mediated modulation. These studies highlight for the first time a role of ultraconserved non-coding RNAs as novel effectors of IL-6 signalling, and the involvement of these RNA genes in the pathogenesis of cholangiocarcinoma. They lay the bases to further investigation of the clinical implications of T-UCR in human cholangiocarcinoma. FundingUniversity of Glasgow.

Dysregulation of vitamin D3 synthesis leads to enhanced cholangiocarcinoma growth

BackgroundCholangiocarcinoma is a deadly biliary tumour with limited treatment strategies. Vitamin (1,25(OH)2D) has anti-proliferative effects on several cancers. Vitamin D3 is synthesized by the enzyme, CYP27B1, and signals via the nuclear vitamin D3 receptor. The enzyme, CYP24A1, degrades vitamin D3. Aims(i) Measure the expression of CYP27B1, CYP24A1, and vitamin D3 receptor in human nonmalignant and cholangiocarcinoma lines and biopsy control or tumour samples; and (ii) evaluate the effects of vitamin D3 on vitamin D3 synthesis and cholangiocarcinoma growth. MethodsIn vitro studies were performed in malignant and nonmalignant cholangiocytes. Vitamin D3 receptor, CYP24 and CYP27 expression was measured in cell lines and biopsy samples. Cell lines were stimulated with vehicle or vitamin D3 from 30min to 48h. Cell viability was assessed by MTS assays and BrdU incorporation. Vitamin D3 receptor, CYP24A1 and CYP27B1 expression was measured in cholangiocarcinoma cells stimulated with vehicle or vitamin D3. ResultsIn cholangiocarcinoma lines and biopsy samples, vitamin D3 receptor and CYP24A1 expression increased compared to controls, whereas CYP27B1 expression was decreased or unchanged. Vitamin D3 induced nuclear translocation of vitamin D3 receptor in cholangiocarcinoma and decreased cholangiocarcinoma growth. ConclusionTreatment with vitamin D3 decreased CYP24A1, whereas CYP27B1 expression increased. Modulation of vitamin D3 synthesis may be important in the management of cholangiocarcinoma.

Molecular pathogenesis of cholangiocarcinoma.

Epidemiological data from the last years show an increasing trend of incidence and mortality of cholangiocarcinoma (CC) worldwide. Many pathophysiologic aspects of this neoplasia are still unknown and need to be fully discovered. However, several progresses were recently made in order to establish the molecular mechanisms involved in the transformation and growth of malignant cholangiocytes. The principal concept that at least seems to be established is that cholangiocarcinogenesis is a multistep cellular process evolving from a normal condition of the epithelial biliary cells through a chronic inflammation status ending with malignant transformation. The bad prognosis related to CC justifies why a better identification of the molecular mechanisms involved in the growth and progression of this cancer is required for the development of effective preventive measures and valid treatment regimens. This Paper describes the scientific progresses made in the last years in defining the molecular pathways implicated in the generation of this devastating disease.

Intraductal papillary neoplasm of the bile duct in liver cirrhosis with hepatocellular carcinoma

A case of intraductal papillary neoplasm of the bile duct (IPNB) arising in a patient with hepatitis B-related liver cirrhosis with hepatocellular carcinoma (HCC) is reported. A 76-year-old man was admitted to our hospital with recurrent HCC. Laboratory data showed that levels of carcinoembryonic antigen and carbohydrate antigen 19-9 were elevated. He died of progressive hepatic failure. At autopsy, in addition to HCCs, an intraductal papillary proliferation of malignant cholangiocytes with fibrovascular cores was found in the dilated large bile ducts in the left lobe, and this papillary carcinoma was associated with an invasive mucinous carcinoma (invasive IPNB). Interestingly, extensive intraductal spread of the cholangiocarcinoma was found from the reactive bile ductular level to the interlobular bile ducts and septal bile ducts and to the large bile ducts in the left lobe. Neural cell adhesion molecule, a hepatic progenitor cell marker, was detected in IPNB cells. It seems possible in this case that hepatic progenitor cells located in reactive bile ductules in liver cirrhosis may have been responsible for the development of the cholangiocarcinoma and HCC, and that the former could have spread in the intrahepatic bile ducts and eventually formed grossly visible IPNB.

Targeting the IL-6 Dependent Phenotype Can Identify Novel Therapies for Cholangiocarcinoma

BackgroundThe need for new therapies for cholangiocarcinoma is highlighted by their poor prognosis and refractoriness to chemotherapy. Increased production of Interleukin-6 promotes cholangiocarcinoma growth and contributes to chemoresistance by activating cell survival mechanisms. We sought to identify biologically active compounds capable of ameliorating the phenotypic effects of IL-6 expression and to explore their potential therapeutic use for cholangiocarcinoma.MethodologyA genomic signature associated with Interleukin-6 expression in Mz-ChA-1 human malignant cholangiocytes was derived. Computational bioinformatics analysis was performed to identify compounds that induced inverse gene changes to the signature. The effect of these compounds on cholangiocarcinoma growth was then experimentally verified in vitro and in vivo. Interactions with other therapeutic agents were evaluated using median effects analysis.Principal FindingsA group of structurally related compounds, nitrendipine, nifedipine and felodipine was identified. All three compounds were cytotoxic to Mz-ChA-1 cells with an IC50 for felodipine of 26 µM, nitrendipine, 44 µM and nifedipine, 15 µM. Similar results were observed in KMCH-1, CC-LP-1 and TFK-1 cholangiocarcinoma cell lines. At a fractional effect of 0.5, all three agents were synergistic with either camptothecin or gemcitabine in Mz-ChA-1 cells in vitro. Co-administration of felodipine and gemcitabine decreased the growth of Mz-ChA-1 cell xenografts in nude athymic mice.ConclusionsComputational bioinformatics analysis of phenotype-based genomic expression can be used to identify therapeutic agents. Using this drug discovery approach based on targeting a defined tumor associated phenotype, we identified compounds with the potential for therapeutic use in cholangiocarcinoma.

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand.

IntroductionCholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.AimsTo determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.ResultsPrimary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.Conclusions1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.

Differential gene expression profiling of cultured neu-transformed versus spontaneously-transformed rat cholangiocytes and of corresponding cholangiocarcinomas

Previously, we described an orthotopic cholangiocarcinoma model based on bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells) compared to high grade malignant erbB-2/neu- transformed BDE1 cholangiocytes (BDEneu cells) into the livers of syngeneic rats, which closely mimics clinical features of early versus advanced stages of the human cancer. We now used gene expression microarray together with quantitative real-time RT-PCR to profile genes differentially expressed in highly tumorigenic BDEneu cells and corresponding tumors compared to less aggressive tumorigenic BDEsp cells and tumors. Genes identified as being commonly overexpressed in parent BDEneu cells, tumors, and in a BDEneu tumor-derived cholangiocarcinoma cell line included Sox17, Krt20, Erbb2, and Sphk1 when respectively compared to BDEsp cells, tumors, and tumor-derived BDEsp cholangiocarcinoma cells. Muc1 was also prominently overexpressed in BDEneu cells and tumor-derived cholangiocarcinoma cells over that expressed in corresponding BDEsp cell lines. Periostin and tenascin-C, which were produced exclusively by cholangiocarcinoma-associated fibroblastic cells, were each significantly overexpressed in BDEneu tumors compared to BDEsp tumors. Interestingly, amphiregulin was representative of a gene found to be significantly underexpressed in vitro in BDEneu cells compared to BDEsp cells, but significantly overexpressed in BDEneu tumors compared to BDEsp tumors, and correlated with BDEneu cholangiocarcinoma progression in vivo. Our data support a unique animal model that recapitulates important molecular features of human cholangiocarcinoma progression, and may serve as a potentially powerful preclinical platform for identifying and rapidly testing novel molecular targeting strategies for cholangiocarcinoma therapy and/or prevention.

Microrna-Dependent Regulation of Dna Methyltransferase-1 and Tumor Suppressor Gene Expression by Interleukin-6 in Human Malignant Cholangiocytes

Although the inflammation-associated cytokine interleukin-6 (IL-6) has been implicated in cholangiocarcinoma growth, the relationship between IL-6 and oncogenic changes is unknown. IL-6 can increase expression of DNA methyltransferase-1 (DNMT-1) and epigenetically regulate the expression of several genes, including microRNAs (miRNAs). DNMT-1 up-regulation occurs in hepatobiliary cancers and is associated with a poor prognosis. To understand the potential regulation of DNMT-1 by IL-6-dependent miRNAs, we examined the expression of a group of miRNAs which have sequence complementarity to the 3'-untranslated region of DNMT-1, namely miR-148a, miR-152, and miR-301. The expression of these miRNAs was decreased in cholangiocarcinoma cells. Moreover, the expression of all three miRNAs was decreased in IL-6-overexpressing malignant cholangiocytes in vitro and in tumor cell xenografts. There was a concomitant decrease in expression of the methylation-sensitive tumor suppressor genes Rassf1a and p16INK4a. Using luciferase reporter constructs, DNMT-1 was verified as a target for miR-148a and miR-152. Precursors to miR-148a and miR-152 decreased DNMT-1 protein expression, increased Rassf1a and p16INK4a expression, and reduced cell proliferation. These data indicate that IL-6 can regulate the activity of DNMT-1 and expression of methylation-dependent tumor suppressor genes by modulation of miR-148a and miR-152, and provide a link between this inflammation-associated cytokine and oncogenesis in cholangiocarcinoma.

Endothelin inhibits cholangiocarcinoma growth by a decrease in the vascular endothelial growth factor expression

Endothelins (ET-1, ET-2, ET-3) are peptides with vasoactive properties interacting with ET(A) and ET(B) receptors. ET-1 inhibits secretin-stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors. The aims of the studies were to evaluate (i) the effect of ET-1 on cholangiocarcinoma growth in Mz-ChA-1 cells and nude mice and (ii) whether ET-1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-2 (VEGFR-2) and VEGFR-3. We determined the expression of ET(A) and ET(B) receptors on normal and malignant (Mz-ChA-1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET-1 on the proliferation and expression of VEGF-A, VEGF-C (regulators of tumour angiogenesis) and its receptors, VEGFR-2 and VEGFR-3, in Mz-ChA-1 cells. In vivo, Mz-ChA-1 cells were injected into the flanks of athymic mice and injections of ET-1 or saline into the tumours were performed daily. The effect of ET-1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF-A and VEGF-C and VEGFR-2 and VEGFR-3 were measured after 73 days. Higher expression of ET(A) and ET(B) was observed in malignant compared with normal cholangiocytes. ET-1 inhibited proliferation and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression of Mz-ChA-1 cells. Chronic ET-1 treatment decreased tumour volume, tumour cell proliferation and VEGF-A and VEGF-C expression but increased apoptosis and collagen tissue deposition compared with controls. Modulation of VEGF-A and VEGF-C (by ET-1) may be important for managing cholangiocarcinoma growth.