Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

Predicting IDH mutation status of intrahepatic cholangiocarcinomas based on contrast-enhanced CT features.

OBJECTIVES: To explore the difference in contrast-enhanced computed tomography (CT) features of intrahepatic cholangiocarcinomas (ICCs) with different isocitrate dehydrogenase (IDH) mutation status. METHODS: Clinicopathological and contrast-enhanced CT features of 78 patients with 78 ICCs were retrospectively analysed and compared based on IDH mutation status. RESULTS: There were 11 ICCs with IDH mutation (11/78, 14.1%) and 67 ICCs without IDH mutation (67/78, 85.9%). IDH-mutated ICCs showed intratumoral artery more often than IDH-wild ICCs (p = 0.023). Most ICCs with IDH mutation showed rim and internal enhancement (10/11, 90.9%), while ICCs without IDH mutation often appeared diffuse (26/67, 38.8%) or with no enhancement (4/67, 6.0%) in the arterial phase (p = 0.009). IDH-mutated ICCs showed significantly higher CT values, enhancement degrees and enhancement ratios in arterial and portal venous phases than IDH-wild ICCs (all p < 0.05). The CT value of tumours in the portal venous phase performed best in distinguishing ICCs with and without IDH mutation, with an area under the curve of 0.798 (p = 0.002). CONCLUSIONS: ICCs with and without IDH mutation differed significantly in arterial enhancement mode, and the tumour enhancement degree on multiphase contrast-enhanced CT was helpful in predicting IDH mutation status. KEY POINTS: • IDH mutation occurred frequently in ICCs. • ICCs with and without IDH mutation differed significantly in arterial enhancement mode. • ICCs with IDH mutation enhanced more than those without IDH mutation. • Enhancement ratio and tumour CT value can predict IDH mutation status.

Activating mutations in PTPN3 promote cholangiocarcinoma cell proliferation and migration and are associated with tumor recurrence in patients.

BACKGROUND & AIMS: The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function. METHODS: We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). RESULTS: Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3. Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3(L232R) and PTPN3(L384H), which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. CONCLUSIONS: Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3. Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

Saturated free fatty acids induce cholangiocyte lipoapoptosis.

UNLABELLED: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth.

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats.

UNLABELLED: Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3',5'-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl(-) /HCO 3- AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct-ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl(-) /HCO 3- AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl(-) /HCO 3- AE2 and ablated secretin-stimulated biliary secretion in these cells. CONCLUSION: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013).

Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.

BACKGROUND & AIMS: Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. METHODS: We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. RESULTS: Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. CONCLUSIONS: We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

Inhibition of Cdc25A suppresses hepato-renal cystogenesis in rodent models of polycystic kidney and liver disease.

BACKGROUND & AIMS: In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. METHODS: Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 (Pkhd1(del2/del2)) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 (Pkd2(ws25/-)) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-)mice; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS: Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared with Pkhd1(del2/del2) mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS: Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.

Portal, but not lobular, macrophages express matrix metalloproteinase-9: association with the ductular reaction and fibrosis in chronic hepatitis C.

BACKGROUND: Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. AIMS: To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. METHODS: Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n = 85). RESULTS: Portal tracts were more densely populated with macrophages (10.5 ± 0.36 macrophages/HPF) than lobules (7.2 ± 0.16 macrophages/HPF, P < 0.001) and macrophages were found in close proximity to the ductular reaction. ≥30% of portal and periductal macrophages expressed MMP-9 and these were significantly associated with increasing stage of fibrosis (rs  = 0.58, 0.68, respectively, both P < 0.001). In contrast, MMP-9(+) macrophages were largely absent in lobular regions and non-diseased liver. Hepatic MMP-9 mRNA levels and gelatinolytic activity were significantly associated with stage of fibrosis (rs  = 0.47, rs  = 0.89, respectively, both P < 0.001). Furthermore, a second distinct CCR2(+) macrophage population was localised to the centrilobular regions and was predominantly absent from portal and periductal areas. CONCLUSIONS: These findings demonstrate significant regional differences in macrophage phenotypes, suggesting that there are at least two populations of liver macrophages. We propose that these populations have distinct contributions to the pathogenesis of chronic HCV-related liver disease.

Oncogenic potential of cyclin kinase subunit-2 in cholangiocarcinoma.

BACKGROUND: Cyclin kinase subunit-2 (Cks2), a member of the human Cks family, plays an important role in the regulation of meiosis and mitosis; and its abnormal expression is usually associated with carcinogenesis. However, its exact functions and molecular mechanisms remain unclear. AIMS: To observe Cks2 expression in cholangiocarcinoma and explore its role in the carcinogenesis of cholangiocarcinoma and possible mechanism. METHODS: Cks2 expression in cholangiocarcinoma was detected with immunostaining and RT-PCR. MTT, colony formation, immunofluorescence, flow cytometry and Western blotting were performed to explore the role of Cks2 in cholangiocarcinoma and possible mechanism. RESULTS: Cks2 was significantly elevated in cholangiocarcinoma tissues and its over-expression was associated with poor differentiation, CA19-9 and poor prognosis. Furthermore, Cks2 down-regulation inhibited cholangiocarcinoma cell proliferation and colony formation in vitro, and the growth of cholangiocarcinoma xenografts in animals; especially, enhanced the sensitivity of cholangiocarcinoma cells to chemotherapy. We further found that Cks2 knockdown induced cholangiocarcinoma cell cycle arrest in G2/M phase through down-regulation of Cyclin A and Cyclin B1 and Bax up-regulation and activation, mitochondrial membrane permeabilization and caspase-3 activation, which resulted in facilitating cholangiocarcinoma apoptosis. CONCLUSIONS: These findings suggest that Cks2 may serve as an independent prognostic factor in patients with cholangiocarcinoma, and play an important role in the carcinogenesis of cholangiocarcinoma by facilitating cell cycle progression and Bax-mediated mitochondrial caspase-dependent apoptosis.

Monoamine oxidase A expression is suppressed in human cholangiocarcinoma via coordinated epigenetic and IL-6-driven events.

The secretion of dopamine and serotonin is increased in cholangiocarcinoma, which has growth-promoting effects. Monoamine oxidase A (MAOA), the degradation enzyme of serotonin and dopamine, is suppressed in cholangiocarcinoma via an unknown mechanism. The aims of this study were to (i) correlate MAOA immunoreactivity with pathophysiological parameters of cholangiocarcinoma, (ii) determine the mechanism by which MAOA expression is suppressed and (iii) evaluate the consequences of restored MAOA expression in cholangiocarcinoma. MAOA expression was assessed in cholangiocarcinoma and nonmalignant controls. The control of MAOA expression by promoter hypermethylation was evaluated and the contribution of interleukin-6 (IL-6) signaling to the suppression of MAOA expression was determined. The effects of MAOA overexpression on cholangiocarcinoma growth and invasion were also assessed. MAOA expression is correlated with differentiation, invasion and survival in cholangiocarcinoma. The MAOA promoter was hypermethylated immediately upstream of the start codon in cholangiocarcinoma samples and cell lines but not in nonmalignant counterparts. IL-6 signaling also decreased MAOA expression via a mechanism independent of hypermethylation, involving the regulation of the balance between SP-1 transcriptional activity and its inhibitor, R1 repressor. Inhibition of both IL-6 signaling and DNA methylation restored MAOA levels to those observed in cholangiocytes. Forced MAOA overexpression inhibited cholangiocarcinoma growth and invasion. MAOA expression is suppressed by the coordinated control of promoter hypermethylation and IL-6 signaling. MAOA may be a useful prognostic marker in the management of cholangiocarcinoma, and therapies designed to increase MAOA expression might prove beneficial in the treatment of cholangiocarcinoma.

Microsomal prostaglandin E synthase-1 inhibits PTEN and promotes experimental cholangiocarcinogenesis and tumor progression.

BACKGROUND & AIMS: Microsomal prostaglandin E synthase-1 (mPGES-1) is a rate-limiting enzyme that is coupled with cyclooxygenase (COX)-2 in the synthesis of prostaglandin E2. Although COX-2 is involved in the development and progression of various human cancers, the role of mPGES-1 in carcinogenesis has not been determined. We investigated the role of mPGES-1 in human cholangiocarcinoma growth. METHODS: We used immunohistochemical analyses to examine the expression of mPGES-1 in formalin-fixed, paraffin-embedded human cholangiocarcinoma tissues. The effects of mPGES-1 on human cholangiocarcinoma cells were determined in vitro and in SCID mice. Immunoblotting and immunoprecipitation assays were performed to determine the levels of PTEN and related signaling molecules in human cholangiocarcinoma cells with overexpression or knockdown of mPGES-1. RESULTS: mPGES-1 is overexpressed in human cholangiocarcinoma tissues. Overexpression of mPGES-1 in human cholangiocarcinoma cells increased tumor cell proliferation, migration, invasion, and colony formation; in contrast, RNA interference knockdown of mPGES-1 inhibited tumor growth parameters. In SCID mice with tumor xenografts, mPGES-1 overexpression accelerated tumor formation and increased tumor weight (P<.01), whereas mPGES-1 knockdown delayed tumor formation and reduced tumor weight (P<.01). mPGES-1 inhibited the expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), leading to activation of the epidermal growth factor/phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathways in cholangiocarcinoma cells. mPGES-1-mediated inhibition of PTEN is regulated through blocking of early growth response-1 sumoylation and binding to the 5'-untranslated region of the PTEN gene. CONCLUSIONS: mPGES-1 promotes experimental cholangiocarcinogenesis and tumor progression by inhibiting PTEN.

The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas.

AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.

Deregulation of Hippo kinase signalling in human hepatic malignancies.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and hepatoblastoma (HB) are the main hepatic malignancies with limited treatment options and high mortality. Recent studies have implicated Hippo kinase pathway in cancer development, but detailed analysis of Hippo kinase signalling in human hepatic malignancies, especially CC and HB, is lacking. METHODS: We investigated Hippo kinase signalling in HCC, CC and HB using cells and patient samples. RESULTS: Increased expression of yes-associated protein (Yap), the downstream effector of the Hippo kinase pathway, was observed in HCC cells, and siRNA-mediated knockdown of Yap resulted in decreased survival of HCC cells. The density-dependent activation of Hippo kinase pathway characteristic of normal cells was not observed in HCC cells and CCLP cells, a cholangiocarcinoma cell line. Immunohistochemistry of Yap in HCC, CC and HB tissues indicated extensive nuclear localization of Yap in majority of tissues. Western blot analysis performed using total cell extracts from patient samples and normal livers showed extensive activation of Yap. Marked induction of Glypican-3, CTGF and Survivin, the three Yap target genes was observed in the tumour samples. Further analysis revealed significant decrease in expression and activity of Lats kinase, the main upstream regulator of Yap. However, no change in activation of Mst-2 kinase, the upstream regulator of Lats kinase was observed. CONCLUSIONS: These data show that Yap induction mediated by inactivation of Lats is observed in hepatic malignancies. These studies highlight Hippo kinase pathway as a novel therapeutic target for hepatic malignancies.

Cyclooxygenase-2 is involved in the up-regulation of matrix metalloproteinase-9 in cholangiocarcinoma induced by tumor necrosis factor-alpha.

Matrix metalloproteinase-9 (MMP-9) is an important enzyme in tumor invasion and metastasis in malignant tumors, including cholangiocarcinoma (CC). Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, was recently reported to induce the up-regulation of MMP-9 in cultured CC cells. We examined whether cyclooxygenase-2 (COX-2) and prostaglandin-E2 (PGE2), another endogenous tumor promoter, are involved in the up-regulation of MMP-9 in CC using CC tissue specimens and a CC cell line, HuCCT-1. MMP-9 and COX-2 were immunohistochemically expressed in 58% and 89% of 110 CC cases, respectively; the expression of MMP-9 and COX-2 was correlated (r = 0.32, P = 0.00072). Using zymography, latent MMP-9 was detectable in all cases and active MMP-9 was detected in 24% of cases of the CC specimens. The TNF-alpha/TNF-receptor 1 (TNF-R1) interaction induced MMP-9 production and activation, as well as COX-2 overexpression and PGE2 production, and increased the migration of CC cells. MMP-9 up-regulation was inhibited by COX inhibitors, antagonists of EP2/4 (receptors of PGE2), and COX-1 and COX-2 siRNAs. Inhibitors of both MMP-9 and MMP-9 siRNA treatment abrogated the increase in the migration of CC cells induced by TNF-alpha. In conclusion, we propose a novel signaling pathway of MMP-9 up-regulation in CC cells such that TNF-alpha induces the activation of COX-2 and PGE2 via TNF-R1 followed by the up-regulation of MMP-9 via the PGE2 (EP2/4) receptor.

Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

Fascin is an actin-binding protein involved in the cell motility. Recently, aberrant expression of fascin in carcinoma cells was reported to participate in their invasive growth in cooperation with proteinases such as matrix metalloproteinases (MMPs). This study examined the participation of fascin in the progression of cholangiocarcinoma (CC) with reference to MMPs and tumor necrosis factor-alpha (TNF-alpha). Expression levels of fascin and MMP2 and 9 were examined immunohistochemically in human non-neoplastic biliary epithelium (13 cases) and CC (87 cases). The relationship between fascin and MMP9-expression levels was examined using two CC cell lines (CCKS-1 and HuCCT1). It was also examined whether or not fascin was involved in TNF-alpha-induced overproduction of MMP9 in CC. Fascin and MMP9 were expressed in 49 and 53% of CC samples, respectively, and the expression of these genes was frequent in intrahepatic CC. Fascin expression was correlated significantly with MMP9 expression. In particular, these two molecules were expressed more intensely at the invasive fronts of CC. Fascin expression was an unfavorable prognostic factor for patients with intrahepatic CC. In vitro studies showed that TNF-alpha could induce the overexpression of fascin and MMP9 in two CC cell lines. A knockdown study of fascin by siRNA showed that TNF-alpha induced the overproduction of fascin, which in turn upregulated MMP9 expression. Overexpression of fascin may have an important function in the progression of CC, and fascin expression might be involved in the signaling pathway in TNF-alpha-dependent production of MMP9 in CC.

Activation-induced cytidine deaminase links bile duct inflammation to human cholangiocarcinoma.

UNLABELLED: Chronic inflammation plays a critical role in oncogenesis in various human organs. Epidemiological studies have demonstrated that patients with primary sclerosing cholangitis have a predisposition to develop cholangiocarcinoma (CC). However, the molecular mechanisms that account for the development of bile duct carcinomas are not well defined. We recently provided evidence that activation-induced cytidine deaminase (AID), a member of the DNA/RNA editing enzyme family, is implicated in human tumorigenesis via its mutagenic activity. We found here that ectopic AID production is induced in response to tumor necrosis factor-alpha (TNF-alpha) stimulation via the IkappaB kinase-dependent nuclear factor-kappaB (NF-kappaB) activation pathway in human cholangiocarcinoma-derived cells. Aberrant expression of AID in biliary cells resulted in the generation of somatic mutations in tumor-related genes, including p53, c-myc, and the promoter region of the INK4A/p16 sequences. In human tissue specimens, real-time reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that AID was increased significantly in 28 of 30 CC tissues (93%), whereas only trace amounts of AID were detected in the normal liver. Immunohistochemistry showed that all of the CC tissue samples examined showed overproduction of endogenous AID protein in cancer cells. Moreover, immunostaining for AID was detectable in 16 of 20 bile epithelia in the tissues underlying primary sclerosing cholangitis. CONCLUSION: The proinflammatory cytokine-induced aberrant production of AID might link bile duct inflammation to an enhanced genetic susceptibility to mutagenesis, leading to cholangiocarcinogenesis.