Human CCA Research Articles

MIR21 Drives Resistance to Heat Shock Protein 90 Inhibition in Cholangiocarcinoma

Background & AimsCholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors.MethodsWe performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice.ResultsCells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21.ConclusionsmiRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents.

Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4+, CD8+ T-cells and in CD56+ NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.

Angiotensin receptor blocker telmisartan inhibits cell proliferation and tumor growth of cholangiocarcinoma through cell cycle arrest

Cholangiocarcinoma (CCA) is at an advanced stage at the time of its diagnosis, and developing a more effective treatment of CCA would be desirable. Angiotensin II type 1 (AT1) receptor blocker (ARB), telmisartan may inhibit cancer cell proliferation, but the mechanisms by which telmisartan affects various cancers remain unknown. In this study, we evaluated the effects of telmisartan on human CCA cells and to assess the expression of microRNAs (miRNAs). We studied the effects of telmisartan on CCA cells using two cell lines, HuCCT-1 and TFK-1. In our experiments, telmisartan inhibited the proliferation of HuCCT-1 and TFK-1 cells. Additionally, telmisartan induced G0/G1 cell cycle arrest via blockade of the G0 to G1 cell cycle transition. Notably, telmisartan did not induce apoptosis in HuCCT-1 cells. This blockade was accompanied by a strong decrease in cell cycle-related protein, especially G1 cyclin, cyclin D1, and its catalytic subumits, Cdk4 and Cdk6. Telmisartan reduced the phosphorylation of EGFR (p-EGFR) and TIMP-1 by using p-RTK and angiogenesis array. Furthermore, miRNA expression was markedly altered by telmisartan in HuCCT-1. Telmisartan inhibits tumor growth in CCA xenograft model in vivo. In conclusion, telmisartan was shown to inhibit human CCA cell proliferation by inducing cell cycle arrest.

Upregulation of TCTP is associated with cholangiocarcinoma progression and metastasis.

In order to investigate the role of translationally-controlled tumor protein (TCTP) in cholangiocarcinoma (CCA) progression and metastasis, TCTP protein staining in paraffin-embedded sections of human CCA tissue samples was examined using immunohistochemistry, and its expression was subsequently compared with clinicopathological parameters. Small interfering RNA (siRNA) targeting TCTP (siTCTP) were transfected into CCA cell lines to evaluate its effects on cellular functions. The proliferation, tumorigenicity and migration abilities of the transfected cells were measured using sulforhodamine B, clonogenic and would healing assays, respectively. The protein levels of TCTP and its associated molecules were evaluated by western blot analysis. Of the 119 individual cases of CCA tissues analyzed, high TCTP scores were significantly correlated with overall metastasis (P=0.044) and a shorter survival time (P<0.001). Multivariate proportional hazards analysis revealed that TCTP is an independent indicator of poor prognosis in CCA (hazard ratio =2.864; P<0.001). siTCTP transfection suppressed CCA cell growth and migration abilities, compared with the control cells (P<0.01). The siTCTP reduced the protein levels of focal adhesion kinase (FAK), phospho-FAK, nuclear factor kappa-light-chain-enhancer of activated B cells and matrix metalloproteinase 9, suggesting potential roles of TCTP in regulating CCA progression and metastasis. In conclusion, the upregulation of TCTP is clinically significant in patients with CCA, serving roles in CCA progression, particularly in cell survival and metastasis. Suppression of TCTP may serve as a potential target in CCA prevention and treatment.

Epigenetic Silencing of miRNA-34a in Human Cholangiocarcinoma via EZH2 and DNA Methylation: Impact on Regulation of Notch Pathway

Aberrant expression and regulation of miRNAs have been implicated in multiple stages of tumorigenic processes. The current study was designed to explore the biological function and epigenetic regulation of miR-34a in human cholangiocarcinoma (CCA). Our data show that the expression of miR-34a is decreased significantly in CCA cells compared with non-neoplastic biliary epithelial cells. Forced overexpression of miR-34a in CCA cells inhibited their proliferation and clonogenic capacity invitro, and suppressed tumor xenograft growth in severe combined immunodeficiency mice. We identified three key components of the Notch pathway, Notch1, Notch2, and Jagged 1, as direct targets of miR-34a. Our further studies show that down-regulation of miR-34a is caused by Enhancer of zeste homolog 2 (EZH2)-mediated H3 lysine 27 trimethylation as well as DNA methylation. Accordingly, treatment with the EZH2 inhibitor, selective S-adenosyl-methionine-competitive small-molecule (GSK126), or the DNA methylation inhibitor, 5-Aza-2'-deoxycytidine, partially restored miR-34a levels in human CCA cells. Immunohistochemical staining and Western blot analyses showed increased EZH2 expression in human CCA tissues and cell lines. We observed that GSK126 significantly reduced CCA cell growth invitro and intrahepatic metastasis invivo. Our findings provide novel evidence that miR-34a expression is silenced epigenetically by EZH2 and DNA methylation, which promotes CCA cell growth through activation of the Notch pathway. Consequently, these signaling cascades may represent potential therapeutic targets for effective treatment of human CCA.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression

Background and aimsCholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. MethodsFXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. ResultsFXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. ConclusionActivation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

TGF-β signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures.

Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-β signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-β1-induced EMT; and (ii) LY2157299, a TGF-β receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. Results: at a dose of 10 μM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30μM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of γ-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 μM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-β signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.

Growth inhibitory effect of β-eudesmol on cholangiocarcinoma cells and its potential suppressive effect on heme oxygenase-1 production, STAT1/3 activation, and NF-κB downregulation.

Cholangiocarcinoma (CCA) is a progressively fatal form of cancer originating from the malignant transformation of hepatic biliary cholangiocytes. The present study reports for the first time in vitro growth inhibitory activities of β-eudesmol, the bioactive sesquiterpenoid present in the rhizome of Atractylodes lancea (Thunb) DC., with respect to its underlying potential effects on heme oxygenase-1 (HO-1) production, STAT1/3 phosphorylation, and NF-κB protein expression in human CCA cell line CL-6. The cytotoxic effect of β-eudesmol on CL-6 cells was evaluated by MTT assay using normal human embryonic fibroblast (OUMS) as a control cell line. Results indicated that β-eudesmol exhibited selective cytotoxicity towards CL-6 compared to OUMS with mean (±SD) IC50 (concentration that inhibits cell growth by 50%) values of 166.75±3.69 and 240.01±16.54μmol/L, respectively. In addition, it also significantly suppressed colony forming and wound healing ability of CL-6 cells in a concentration-dependent manner. Western blot analysis indicated that β-eudesmol treatment resulted in significant suppression of HO-1 production in CL-6 cells. Its inhibitory effects on the phosphorylation of STAT1/3 proteins and expression of NF-κB (p65 and p50) proteins were concentration-dependent. Taken together, these results suggest that β-eudesmol exerts significant growth inhibitory activity on CL-6 cells that may be linked to its inhibitory effect on the production of HO-1, phosphorylation of STAT1/3, and expression of major NF-κB proteins.

Platelet-derived growth factor regulates YAP transcriptional activity via Src family kinase dependent tyrosine phosphorylation.

The Hippo pathway effector YAP is implicated in the pathogenesis of cholangiocarcinoma (CCA). The Hippo pathway relies on signaling cross talk for its regulation. Given the importance of platelet derived growth factor receptor (PDGFR) signaling in CCA biology, our aim was to examine potential YAP regulation by PDGFR. We employed human and mouse CCA specimens and cell lines for these studies. Initially, we confirmed upregulation of PDGFRβ and PDGFR ligands in human and mouse CCA specimens and cell lines. YAP, a transcriptional co-activator, was localized to the nucleus in human CCA specimens and a cell line, as well as patient derived xenografts (PDX). PDGFR pharmacologic inhibition led to a redistribution of YAP from the nucleus to cytosol and downregulation of YAP target genes in a human CCA cell line. siRNA silencing of PDGFR-β similarly downregulated YAP target genes. YAP activation (nuclear localization and target gene expression) was regulated by Src family kinases (SFKs) downstream of PDGFR. SFK activity resulted in phosphorylation of YAP on tyrosine357 (YAPY357 ). The importance of YAPY357 phosphorylation in regulating YAP activation was confirmed utilizing the SB-1 cell line, a mouse cell line expressing YAP S127A precluding canonical serine phosphorylation. PDGFR inhibition decreased cellular abundance of the survival protein Mcl-1, a known YAP target gene, and accordingly increased cell death in CCA cells in vitro and in vivo. These preclinical data demonstrate that a PDGFR-SFK cascade regulates YAP activation via tyrosine phosphorylation in CCA. Inhibiting this cascade may provide a viable therapeutic strategy for this human malignancy.

Elevated levels of circulating osteopontin are associated with a poor survival after resection of cholangiocarcinoma

Cholangiocarcinoma (CCA) represents a primary hepatic malignancy with incidence and mortality rising globally. Surgical treatment has remained the only potentially curative treatment option, but it is still unclear which patients benefit most from extended liver surgery, highlighting the need for new pre-operative stratification strategies. Osteopontin is a secreted extracellular glyco-phosphoprotein that has been associated with inflammation, metabolic disorders and cancer. Here, we examined the potential of circulating osteopontin serum levels as a diagnostic or prognostic biomarker in patients with CCA undergoing extended liver surgery. Osteopontin expression levels were analysed in human and murine CCA tumour samples, using semi-quantitative reverse transcriptase PCR and immunohistochemistry. Osteopontin serum concentrations were measured by enzyme-linked immunosorbent assay in 107 patients with CCA undergoing elective tumour resection as well as 55 healthy controls. Results were correlated with clinical data. Correlating with an upregulation in CCA tumour cells and the tumour stroma, serum levels of osteopontin were elevated in patients with cholangiocarcinoma compared to healthy controls and patients with primary sclerosing cholangitis. Importantly, pre- and postoperative elevations of osteopontin showed a striking association with poor postoperative survival. Serum osteopontin concentrations represent a promising prognostic biomarker in patients resectable CCA which could help to guide preoperative treatment decisions and to identify patients that will particularly benefit from extended liver surgery. Lay summary: Extended liver surgery is the only potentially curative treatment for patients with cholangiocarcinoma (CCA/biliary cancer), but it is currently unclear which patients benefit most from surgery. Detecting serum levels of osteopontin - a specific secreted glycoprotein involved in multiple human diseases - in CCA patients might help to identify those patients that particularly benefit from tumour resection.

Histone deacetylase 3 overexpression in human cholangiocarcinoma and promotion of cell growth via apoptosis inhibition

Histone deacetylase 3 (HDAC3) has an oncogenic role in apoptosis and contributes to the proliferation of cancer cells. MI192 is a novel HDAC3-specific inhibitor that displays antitumor activity in many cancer cell lines. However, the role of HDAC3 and the antitumor activity of its inhibitor MI192 are not known in cholangiocarcinoma (CCA). The present study aims to identify the target of MI192 in CCA as well as evaluate its therapeutic efficacy. CCK8 and colony formation assays showed that HDAC3 overexpression promotes proliferation in CCA cell lines. HDAC3 knockdown or treatment with MI192 decreased CCA cell growth and increased caspase-dependent apoptosis, while apoptosis was partially rescued by HDAC3 overexpression. We demonstrated that MI192 can inhibit the deacetylation activity of HDAC3 and its downstream targets in vitro, and MI192 inhibited xenograft tumor growth in vivo. Immunochemistry showed that HDAC3 was upregulated in CCA tissues compared with adjacent normal tissues, and this was correlated with reduced patient survival. Taken together, these results demonstrate for the first time that MI192 targets HDAC3 and induces apoptosis in human CCA cells. MI192 therefore shows the potential as a new drug candidate for CCA therapy.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

Anticholangiocarcinoma activity and toxicity of the Kaempferia galanga Linn. Rhizome ethanolic extract

BackgroundCholangiocarcinoma (CCA) is an important public health problem in several tropical and subtropical parts of the world particularly Thailand. Chemotherapy of CCA is largely ineffective and discovery and development of effective alternative drugs is urgently needed. The objective of the study was to confirm the anti-CCA potential as well as toxicity of the crude extract of Kaempferia galangal Linn. (rhizome) both in vitro and in animal models.MethodsThe ethanolic extract of K. galanga Linn. rhizome, ethyl-p-methoxycinnamate (EPMC) and 5-fluorouracil (5-FU) were evaluated for their cytotoxic activities against CCA cell line (CL-6) using MTT cell proliferation assay. Acute and subacute toxicity of the extract were evaluated in ICR (Imprinting Control Region) mice according to the OECD (International Organization for Economic Co-operation and Development) Guideline. Anti-CCA activity was evaluated in CCA- xenografted nude mice.ResultsResults of cytotoxicity test showed moderate activity of the extract and EPMC with median (95% confidence interval: 95% CI) 50% inhibitory concentration (IC50) of 64.2 (57.76–72.11) and 49.19 (48.16–52.29) μg/ml, respectively. The IC50 of 5-FU was 107.1 (103.53–109.64) μg/ml. The selectivity index (SI) values for the extract, EPMC and 5-FU against human normal cell line (OUMS) and cancer cell line (CL-6) were 2.2, 2.09 and 1.31, respectively. Toxicity testing revealed no overt toxic effect up to the maximum single oral dose of 5000 mg/kg body weight and up to daily dose of 1000 mg/kg body weight for 30 days. The extract at the maximum tolerated dose level of 1000 mg/kg body weight for 30 days exhibited promising anti-CCA activity in CL6-xenografted nude mice as determined by inhibitory activity on tumor growth (58.41%) and lung metastasis (33.3%), as well as prolongation of survival time (62 days).ConclusionThe K. galangal Linn. rhizome extract and its bioactive compound EPMC exhibited moderate cytotoxic activity against human CCA tumor (CL-6) cell line. Results of toxicity testing suggest that the extract was well tolerated up to the maximum single oral dose of 5000 mg/kg body weight and daily dose of 1000 mg/kg body weight for 30 days. The extract exhibited promising anti-CCA activity in CL6-xenografed nude mice as determined by significant inhibitory activity on tumor growth and lung metastasis, as well as prolongation of survival time.

MART-10 represses cholangiocarcinoma cell growth and high vitamin D receptor expression indicates better prognosis for cholangiocarcinoma

Cholangiocarcinoma (CCA) is a devastating disease due to no effective treatments available. Since the non-mineral functions of vitamin D emerges, 1α,25(OH)2D3, the active form of vitamin D, has been applied in anti-cancer researches. In this study, we demonstrated that both the 1α,25(OH)2D3 analog, MART-10, and 1α,25(OH)2D3 possessed anti-growth effect on human CCA cells with MART-10 much more potent than 1α,25(OH)2D3. The growth inhibition of both drugs were mediated by induction of G0/G1 cell cycle arrest through upregulation of p27 and downregulation of CDK4, CDK6, and cyclin D3. Human neutrophil gelatinase associated lipocalin (NGAL) was found to be involved in 1α,25(OH)2D3 and MART-10 meditated growth inhibition for CCA as knockdown of NGAL decreased Ki-67 expression in SNU308 cells and rendered SNU308 cells less responsive to 1α,25(OH)2D3 and MART-10 treatment. Vitamin D receptor (VDR) knockdown partly abolished MART-10-induced inhibition of NGAL and cell growth in SNU308 cells. The xenograft animal study demonstrated MART-10 could effectively repressed CCA growth in vivo without inducing obvious side effects. The IHC examination of human CCA specimen for VDR revealed that higher VDR expression was linked with better prognosis. Collectively, our results suggest that MART-10 could be a promising regimen for CCA treatment.

SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. SOX17 expression/function was evaluated along the differentiation of human induced pluripotent stem cells (iPSC) into cholangiocytes, in the dedifferentiation process of normal human cholangiocytes (NHC) in culture and in cholangiocarcinogenesis. Lentiviruses for SOX17 overexpression or knockdown were used. Gene expression and DNA methylation profiling were performed. SOX17 expression is induced in the last stage of cholangiocyte differentiation from iPSC and regulates the acquisition of biliary markers. SOX17 becomes downregulated in NHC undergoing dedifferentiation; experimental SOX17 knockdown in differentiated NHC downregulated biliary markers and promoted baseline and Wnt-dependent proliferation. SOX17 expression is lower in human CCA than in healthy tissue, which correlates with worse survival after tumor resection. In CCA cells, SOX17 overexpression decreased their tumorigenic capacity in murine xenograft models, which was related to increased oxidative stress and apoptosis. In contrast, SOX17 overexpression in NHC did not affect their survival but inhibited their baseline proliferation. In CCA cells, SOX17 inhibited migration, anchorage-independent growth and Wnt/β-catenin-dependent proliferation, and restored the expression of biliary markers and primary cilium length. In human CCA, SOX17 promoter was found hypermethylated and its expression inversely correlates with the methylation grade. In NHC, Wnt3a decreased SOX17 expression in a DNMT-dependent manner, whereas in CCA, DNMT1 inhibition or silencing upregulated SOX17. SOX17 regulates the differentiation and maintenance of the biliary phenotype and functions as a tumor suppressor for CCA, being a potential prognostic marker and a promising therapeutic target. Understanding the molecular mechanisms involved in the pathogenesis of CCA is key in finding new valuable diagnostic and prognostic biomarkers, as well as therapeutic targets. This study provides evidence that SOX17 regulates the differentiation and maintenance of the biliary phenotype, and its downregulation promotes their tumorigenic transformation. SOX17 acts as a tumor suppressor in CCA and its genetic, molecular and/or pharmacological restoration may represent a new promising therapeutic strategy. Moreover, SOX17 expression correlates with the outcome of patients after tumor resection, being a potential prognostic biomarker.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

β-caryophyllene and low-doses of doxorubicin against liver cancer cells: a “metronomic chemotherapy”

Cholangiocarcinoma and hepatocellular carcinoma are primary liver cancers, both representing a growing challenge due to their increasing morbidity and mortality. A “metronomic chemotherapy”, consisting of the repeated administration of low and/or continuous doses of anti-neoplastic drugs, represents an alternative approach to the standard chemotherapy [1]. Numerous natural substances exhibited in vitro chemosensitizing features: in particular, the natural sesquiterpene β-caryophyllene (CRY) has been proved to increase the cytotoxicity of doxorubicin (DOXO) in leukemic cells [2]. Hence, our aim has been to evaluate the ability of CRY to enhance the efficacy of low-dose DOXO in human liver cancer cells, by applying a metronomic protocol. To this end, human liver HepG2 and CCA cells have been used as models of hepatocellular carcinoma and cholangiocarcinoma. The metronomic protocol was based on a 2h low-time exposition to the test substances, followed by 72h incubation for restoring. This scheduling has been applied 3 times and cytotoxicity was measured by MTT assay. Both the substances alone (CRY 1-100 μg/ml; DOXO 1-500 μg/ml) and the combination of DOXO with a nontoxic concentration of CRY were assessed. We found that the repeated treatments with low concentrations produced a significant potentiation (about 30 %) of DOXO cytotoxicity in HepG2. The combination with CRY increased the DOXO activity, reaching a 70 % inhibition of cell viability at 50 μg/ml after 2 repeated treatments. Similar effects were found in CCA, although repeated treatments induced no additional potentiation. These results highlight a possible role of CRY as a chemosensitizing agent for DOXO-based chemotherapy of liver cancer.

Metformin Exerts Antiproliferative and Anti-metastatic Effects Against Cholangiocarcinoma Cells by Targeting STAT3 and NF-ĸB

Cholangiocarcinoma (CCA) is an aggressive cancer for which standard treatments are still ineffective. This study demonstrated the antiproliferative and anti-metastatic activity of metformin, an anti-diabetic drug, in CCA cells. Cell proliferation, migration/invasion and anoikis resistance were determined. The underlying mechanisms were identified using western blotting and immunocytofluorescence. Metformin significantly suppressed proliferation of CCA cells in a dose- and time-dependent manner, regardless of glucose present in the medium. A low dose of metformin significantly increased anoikis and inhibited migration/ invasion of CCA cells that was in concert with the decrease of vimentin, matrix metalloproteinase (MMP)-2 and -7. Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) by phosphorylation together with suppression of nuclear translocation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-ĸB) were the underlying mechanisms for these effects. Metformin is a potent antiproliferative and anti-metastatic agent against human CCA cells. These findings encourage the repurposing of metformin in clinical trials to improve CCA treatment.

In Vitro and In Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA.

Imbalanced adaptive responses associated with microsatellite instability in cholangiocarcinoma.

The adaptive response of the genome protection mechanism occurs in cells when exposed to genotoxic stress due to the overproduction of free radicals via inflammation and infection. In such circumstances, cells attempt to maintain health via several genome protection mechanisms. However, evidence is increasing that this adaptive response may have deleterious effect; a reduction of antioxidant enzymes and/or imbalance in the DNA repair system generates microsatellite instability (MSI), which has procarcinogenic implications. Therefore, the present study hypothesized that MSI caused by imbalanced responses of antioxidant enzymes and/or DNA repair enzymes as a result of oxidative/nitrative stress arising from the inflammatory response is involved in liver fluke-associated cholangiocarcinogenesis. The present study investigated this hypothesis by identifying the expression patterns of antioxidant enzymes, including superoxide dismutase 2 (SOD2) and catalase (CAT), and DNA repair enzymes, including alkyladenine DNA glycosylase (AAG), apurinic endonuclease (APE) and DNA polymerase β (DNA pol β). In addition, the activities of the antioxidant enzymes, SOD2 and CAT, were examined in human cholangiocarcinoma (CCA) tissues using immunohistochemical staining. MSI was also analyzed in human CCA tissues. The resulting data demonstrated that the expression levels of the SOD2 and CAT enzymes decreased. The activities of SOD2 and CAT decreased significantly in the CCA tissues, compared with the hepatic tissue of cadaveric donors. In the DNA repairing enzymes, it was found that the expression levels of AAG and DNA pol β enzymes increased, whereas the expression of APE decreased. In addition, it was found that MSI-high was present in 69% of patients, whereas MSI-low was present in 31% of patients, with no patients classified as having microsatellite stability. In the patients, a MSI-high was correlated with poor prognosis, indicated by a shorter survival rate. These results indicated that the reduction of antioxidant enzymes and adaptive imbalance of base excision repair enzymes in human CCA caused MSI, and may be associated with the progression of cancer.