Gábor Lendvai

The presence of autophagy has been indicated in cholangiocarcinoma (CC), which disease has poor prognosis and limited treatment options. Recently, CC has been classified by anatomical localization as intrahepatic (iCC), perihilar (pCC) and distal (dCC), showing different clinical and molecular characteristics. Thus, our aim was to compare autophagy activity in CC samples resected from different anatomical locations. Further, we investigated whether autophagy could be modulated in cell lines originated from iCC and extrahepatic CC (eCC) following the treatments with autophagy inhibitory and inducing agents. Tissue microarrays were prepared from 70 CC (28 iCC, 19 pCC and 23 dCC), 31 adjacent non-tumorous and 9 hepatocellular carcinoma (HCC) samples. Autophagy markers LC3, p62 and Beclin1 as well as proliferation marker Ki-67 were monitored by immunohistochemistry and were associated with patients’ survival. Modulation of autophagy was investigated in cell lines originated from iCC (HuH-28), eCC (TFK-1) and HCC (HepG2) by treating the cells with chloroquine (CQ) for inhibition and with Rapamycin, 5-Fluorouracil (5-FU) and Sorafenib for induction of autophagy. Our results indicated an inhibited autophagy in iCC and pCC tumor tissues, whereas active autophagy seemed to occur in dCC, especially in samples displaying low Ki-67 index. Additionally, low level of Beclin1 and high level of Ki-67 were associated with poor overall survival in dCC, suggesting the prognostic role of these proteins in dCC. Beside a baseline autophagy detected in each cell line, Rapamycin and 5-FU induced autophagy in iCC and HepG2 cell lines, Sorafenib in iCC cells. A chemotherapy agent in combination with CQ decreased IC50 effectively in the cell lines where basal and/or induced autophagy were present. In conclusion, we revealed differences in the autophagy activities of CC tissues and cell lines originated from different anatomical locations, which might influence patients’ treatment. Our results also suggest a prognostic role of Beclin1 and Ki-67 in dCC.

The unique character of selenium compounds, including sodium selenite and Se-methylselenocysteine (MSC), is that they exert cytotoxic effects on neoplastic cells, providing a great potential for treating cancer cells being highly resistant to cytostatic drugs. However, selenium treatment may affect microRNA (miRNA) expression as the pattern of circulating miRNAs changed in a placebo-controlled selenium supplement study. This necessitates exploring possible changes in the expression profiles of miRNAs. For this, miRNAs being critical for liver function were selected and their expression was measured in hepatocellular carcinoma (HLE and HLF) and cholangiocarcinoma cell lines (TFK-1 and HuH-28) using individual TaqMan MicroRNA Assays following selenite or MSC treatments. For establishing tolerable concentrations, IC₅₀ values were determined by performing SRB proliferation assays. The results revealed much lower IC₅₀ values for selenite (from 2.7 to 11.3 μM) compared to MSC (from 79.5 to 322.6 μM). The treatments resulted in cell line-dependent miRNA expression patterns, with all miRNAs found to show fold change differences; however, only a few of these changes were statistically different in treated cells compared to untreated cells below IC₅₀. Namely, miR-199a in HLF, miR-143 in TFK-1 upon MSC treatment, miR-210 in HLF and TFK-1, miR-22, -24, -122, −143 in HLF upon selenite treatment. Fold change differences revealed that miR-122 with both selenium compounds, miR-199a with MSC and miR-22 with selenite were affected. The miRNAs showing minimal alterations included miR-125b and miR-194. In conclusion, our results revealed moderately altered miRNA expression in the cell lines (less alterations following MSC treatment), being miR-122, −199a the most affected and miR-125b, -194 the least altered miRNAs upon selenium treatment.

Although the role of autophagy has been implicated in several forms of chronic hepatitis, it is still not fully understood. Active autophagy eliminates damaged molecules and organelles (such as mitochondria) by lysosomal degradation. In the present study, we aimed to examine and compare autophagy activity in chronic hepatitis C (CHC) and autoimmune hepatitis (AIH) by detecting the expression of autophagy (LC3 and p62) and mitochondrium-related (TOMM20) proteins, as well as the levels of selected microRNAs (miR-101, -155, -204 and − 224) known to be involved in the regulation of autophagy. In addition, the expression levels were related to pathohistological parameters. Liver biopsy samples, including 45 CHC and 18 AIH cases, were immunohistochemically stained for LC3, p62 and TOMM20 and the expression of miRNAs was determined using real-time PCR. We found elevated LC3 and p62 in AIH samples as compared with CHC ones, indicating an activated autophagy that is impaired in AIH as no degradation of p62 seemed to occur. Moreover, p62 showed strong correlation with necroinflammatory grades in the AIH group. The observed elevated levels of TOMM20 and p62 suggest a less efficient elimination of damaged mitochondria in AIH as opposed to CHC, in which autophagy seems to have a more active function. The level of miR-101 was increased in case of CHC as compared with AIH, however, miR-155, -204 and 224 resulted in no expressional. Furthermore, miR-224 level correlated with steatosis and miR-155 expression with fibrosis stage in CHC. In conclusion, dissimilar autophagic activity was observed in CHC and AIH, suggesting a close association between impaired autophagy and severity of necroinflammation. This impairment may not be regulated by the analyzed miRNAs. Nevertheless, miR-224 and − 155 seem to be associated with CHC progression.

Cholangiocarcinoma (CC) is the second most common tumor of the liver, originating from the biliary system with increasing incidence and mortality worldwide. Several new classifications review the significance of tumor localization, site of origin, proliferation and biomarkers in the intrahepatic, perihilar and distal forms of the lesion. Based on growth pattern mass-forming, periductal-infiltrating, intraductal, undefined and mixed types are differentiated. There are further subclassifications which are applied for the histological features, in particular for intrahepatic CC. Recognition of the precursors and early lesions of CC including biliary intraepithelial neoplasia (BilIN), intraductal papillary neoplasm of the bile ducts (IPNB), biliary mucinous cystic neoplasm (MCNB) and the candidate precursors, such as bile duct adenoma and von Meyenburg complex is of increasing significance. In addition to the previously used biliary markers detected by immunohistochemistry, several new markers have been added to the differentiation of both the benign and malignant lesions, which can be used to aid in the subclassification in association with the outcome of CC. Major aspects of biliary carcinogenesis have been revealed, yet, the exact way of this diverse process is still unclear. The factors contributing to molecular cholangiocarcinogenesis include various risk factors, different anatomical localizations, multiple cellular origins, genetic and epigenetic alterations, tumor microenvironment, heterogeneity and clonal evolution. Driver mutations have been identified, implying that they are optimal candidates for targeted therapy. The most promising therapeutic candidates have entered clinical trials.

The liver disease focal nodular hyperplasia (FNH) has several histological features that resemble hepatic cirrhosis. Since cirrhosis may develop further into hepatocellular carcinoma (HCC) contrary to FNH, the aim of the present study was to identify microRNAs (miRNA), which, by their altered expression levels, may be associated with the benign, tumor-like nature of FNH. Altogether 106 surgically removed formalin-fixed paraffin-embedded liver samples were selected, including 22 FNH, 45 cirrhosis, 24 HCC and 15 normal liver tissues. Etiology of the cases of cirrhosis and HCC includes hepatitis C and alcoholism and the HCC cases developed in cirrhotic livers. Relative expression levels of 14 miRNAs were determined using TaqMan MicroRNA Assays. In comparison to normal liver, the levels of miR-34a and miR-224 were elevated not only in FNH but also in cirrhosis and HCC, while the expression of miR-17-5p, miR-18a and miR-210 was decreased in FNH. Further, the levels of miR-21 and miR-222 were increased in cirrhosis and HCC but were decreased in FNH and the expression of miR-17-5p, miR-18a, miR-195 and miR-210 was decreased in FNH as compared with cirrhosis and/or HCC. In conclusion, the elevation of miR-34a and miR-224 may be associated with both benign and malignant proliferative processes, nevertheless the increased expression of oncomiRs miR-21 and miR-222 in cirrhosis and HCC but not in FNH may be related to malignant processes of the liver. The decreased levels of miR-18a, miR-195 and miR-210 may further differentiate FNH from cirrhosis, reflecting the different pathogenesis of these two entities contrary to some histologically similar features.

Data discussed in recent reviews demonstrated that dysregulation of microRNA (miRNA) expression profiles occurs during cervical carcinogenesis and characteristic up- or downregulation of certain miRNAs might be used as biomarkers. The majority of altered miRNAs, however were found to be inconsistent upon comparison with cancerous and normal cervical epithelia in the discussed studies due to several reasons. The results obtained in this present review suggest the need for further investigations on miRNAs on larger sample sizes in order to indicate sensitivity and specificity by means of well defined, “unified” methods. In addition, obtaining further data on the clinical course and outcome of patients in comparison to the dysregulation of miRNA expression profile could turn miRNAs into prognostic and/or progression markers. Inhibition of overexpressed miRNAs, as suggested by some authors, might even serve as target for cancer therapy.

AIMS\ud \ud The more differentiated fetal component of hepatoblastoma (HB) is characterized by increased expression of tight junction (TJ) proteins claudin-1 and -2 when compared with embryonal component. Expression patterns of the recently identified TJ protein tricellulin and the epigenetic regulator enzyme EZH2 were investigated in epithelial subtypes of HB and related to survival.\ud \ud METHODS AND RESULTS\ud \ud Twenty-one cases of epithelial HBs subtyped as pure fetal (n = 12) and embryonal/fetal (n = 9), along with 16 non-tumorous samples from surrounding liver, were analysed by immunohistochemistry for tricellulin, β-catenin and EZH2 expression. No significant differences were revealed in overall survival between fetal and embryonal/fetal types of HBs. The fetal component, however, showed considerably increased tricellulin expression while the embryonal component displayed significantly increased nuclear EZH2 positivity, in comparison to other epithelial subtypes and non-tumorous surrounding hepatocytes. Strong nuclear β-catenin staining was notably more frequent in embryonal than in fetal types. High tricellulin expression was associated with significantly increased overall survival (P = 0.03), while elevated EZH2 expression was linked to the presence of distant metastases (P = 0.013).\ud \ud CONCLUSIONS\ud \ud Our data indicate that patients with treated HBs showing high expression of tricellulin have significantly better overall survival, independent of histological subtype. Increased nuclear expression of EZH2 was associated with the presence of distant metastases

Hepatoblastoma (HB) is the most common primary liver cancer in childhood. The fetal and mixed embryonal/fetal epithelial subtypes of HB differ not only in grade of differentiation but probably also in prognosis. We aimed to determine microRNA (miRNA) expression patterns of the main subtypes of epithelial HBs to reveal differences and relate them to survival. We studied 20 cases of epithelial HB, subtyped as pure fetal (n = 12) or embryonal/fetal (n = 8). Tissues were sampled according to subtype to arrive at 15 purely fetal and eight purely embryonal samples (n = 8) and 15 samples of non-tumorous surrounding liver (SL). Relative expression of miR-17-5p, miR-18a, miR-21, miR-34a, miR-96, miR-122, miR-181a, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, and miR-224 was determined by TaqMan MicroRNA Assays applying miR-140 as reference. A higher level of miR-18a (p < 0.01) was found in embryonal samples than in fetal samples. Lower miR-17-5p, miR-195, miR-210, miR-214, and higher miR-221 levels were detected in fetal samples (p < 0.02) in comparison with SL samples, whereas a lower miR-122 level was observed in embryonal samples (p < 0.003). Histological subtype did not correlate with survival; however, high miR-21, low miR-222, and low miR-224 levels proved to be independently prognostic for HB with significantly increased overall survival (p < 0.03). The fetal and embryonal components of epithelial HB, as well as SL, revealed different miRNA expression patterns. Furthermore, miR-21, miR-222, and miR-224 levels predict overall survival of HB patients regardless of epithelial subtype.

Background and Aim: Management of hepatitis C virus (HCV) recurrence is a major challenge after liver transplantation. Significant dysregulated expression of HCV receptors (i.e. claudin‐1, occludin, tetraspanin CD81, scavenger receptor type B1) has been shown recently during HCV infection. This might facilitate hepatocytic entry and reinfection of HCV. MicroRNAs (miRs) play role in the regulation of gene expression. We aimed to characterize miR expression profiles related to HCV infection and antiviral therapy in adult liver transplant recipients, with special emphasis on miRs predicted to target HCV receptors.Methods: Twenty‐eight adult liver transplant recipients were enrolled in the study. Paired biopsies were obtained at the time of HCV recurrence and at the end of antiviral treatment. MiRs for HCV receptors were selected using target prediction software. Expression levels of miR‐21, miR‐23a miR‐34a, miR‐96, miR‐99a*, miR‐122, miR‐125b, miR‐181a‐2*, miR‐194, miR‐195, miR‐217, miR‐221, and miR‐224 were determined by reverse transcription–quantitative polymerase chain reaction.Results: miR‐99a* and miR‐224 expressions were increased in HCV recurrence samples, while miR‐21 and miR‐194 were decreased in comparison to normal liver tissue. Increased expressions of miR‐221, miR‐224, and miR‐217 were observed in samples taken after antiviral therapy when compared with HCV recurrence samples. High HCV titer at recurrence was associated with higher level of miR‐122.Conclusions: Samples at recurrence of HCV and after antiviral therapy revealed distinct HCV‐related miR expression profiles, with significant dysregulation of those miRNAs potentially targeting mRNAs of HCV receptors. In particular, miR‐194 and miR‐21 might be involved in the regulation of HCV receptor proteins' expression during HCV infection and antiviral therapy.

The details of molecular alterations occurring during hepatocarcinogenesis have not been revealed yet. Nevertheless, it is known that microRNAs (miRNA), these short RNA molecules regulating gene expression mainly in a negative way, are also involved in this process. Altered miRNA expression levels are present in liver diseases when compared with normal liver tissue, and the observed alterations depend mainly on which is more advantegous for the disease: activation or inhibition of the genes (e.g. oncogenes or tumor suppressor genes) regulated by the altered miRNAs. The miRNA expression pattern described in hepatocellular carcinoma seems to differ the most from that found in the normal liver; however, remarkable alterations at miRNA levels have been published in early stages of hepatic tumor progression such as fibrosis and chronic hepatitis. For example, the expression of miR-21, miR-221, miR-222 and miR-199a showing characteristic alterations in hepatocellular carcinoma also displayed deregulated expressions in these two early stages. The liver characteristic miRNA, miR-122, usually exhibits a decreased expression level upon liver injury as well as miR-122 expression tends to decrease as hepatic carcinogenesis progresses. Besides, miR-122 enhances the replication of hepatitis C virus and the initial low or high level of miR-122 seems to influence the efficiency of interferon therapy. Recently, statistically significant differences have been detected in the expression of several miRNAs being present in the serum of patients with chronic hepatitis, chirrhosis and hepatocellular carcinoma when compared with normal controls. It suggests that serum miRNAs could be potential biomarkers. In this article, the major and recent alterations of microRNA expression patterns in stages of hepatocarcinogenesis such as fibrosis, viral infections (hepatitis), cirrhosis and hepatocellular carcinoma are summarized. Orv. Hetil., 2012, 153, 978–989.