The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or nontumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.

Hepatic lipid deposition and inflammation represent risk factors for hepatocellular carcinoma (HCC). The mRNA-binding protein tristetraprolin (TTP, gene name ZFP36) has been suggested as a tumor suppressor in several malignancies, but it increases insulin resistance. The aim of this study was to elucidate the role of TTP in hepatocarcinogenesis and HCC progression. Employing liver-specific TTP-knockout (lsTtp-KO) mice in the diethylnitrosamine (DEN) hepatocarcinogenesis model, we observed a significantly reduced tumor burden compared to wild-type animals. Upon short-term DEN treatment, modelling early inflammatory processes in hepatocarcinogenesis, lsTtp-KO mice exhibited a reduced monocyte/macrophage ratio as compared to wild-type mice. While short-term DEN strongly induced an abundance of saturated and poly-unsaturated hepatic fatty acids, lsTtp-KO mice did not show these changes. These findings suggested anti-carcinogenic actions of TTP deletion due to effects on inflammation and metabolism. Interestingly, though, investigating effects of TTP on different hallmarks of cancer suggested tumor-suppressing actions: TTP inhibited proliferation, attenuated migration, and slightly increased chemosensitivity. In line with a tumor-suppressing activity, we observed a reduced expression of several oncogenes in TTP-overexpressing cells. Accordingly, ZFP36 expression was downregulated in tumor tissues in three large human data sets. Taken together, this study suggests that hepatocytic TTP promotes hepatocarcinogenesis, while it shows tumor-suppressive actions during hepatic tumor progression.

Overexpression of the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2/IGF2BP2) has been described in different cancer types. Gallbladder carcinoma (GBC) is a rare but highly aggressive cancer entity with late clinical detection and poor prognosis.The aim of this study was to investigate the role of IMP2 in human GBC.Tissue microarrays (TMAs) of an international multi-center GBC sample collection from n = 483 patients were analyzed by immunohistochemistry. IMP2 immunoreactivity was found in 74.3% of the tumor samples on TMA, of which 14.0% showed strong and 86.0% low staining intensity. 72.4% of the tumor samples were IMP1 positive, but IMP1 showed lower expression in tumor tissue compared to control tissues. IMP3 immunoreactivity was observed in 92.7% of all tumors, of which 53.6% revealed strong IMP3 expression. Kaplan-Meier analysis linked high IMP2 expression to shorter survival time (p = 0.033), whereas neither IMP1 nor IMP3 expression was linked to a decreased survival time. Eight different human biliary tract cancer (BTC) cell lines were evaluated for tumor growth kinetics in mouse xenografts. Cell lines with high IMP2 expression levels showed the fastest increase in tumor volumes in murine xenografts. Furthermore, IMP2 expression in these cells correlated with the generation of reactive oxygen species (ROS) and RAC1 expression in BTC cells, suggesting RAC1-induced ROS generation as a potential mechanism of IMP2-promoted progression of GBC.In conclusion, IMP2 is frequently overexpressed in GBC and significantly associated with poor prognosis and growth rates in vivo. IMP2 might therefore represent a new target for the treatment of advanced GBC.

Recent evidence published in the Journal of Hepatology shed light on the fate of Kupffer cells (KCs) during hepatic inflammation. The study by Borst et al. elegantly demonstrated that KCs are rapidly depleted and then replaced by monocyte-derived macrophages in the course of viral hepatitis. 1 This report is quite in contrast to the dogma that KCs, as resident tissue macrophages, are self-maintaining cells. Similar observations were reported for hepatic inflammation induced by paracetamol, CCl 4 , or bacterial infection. 2-4 All of these studies show a temporary decline of KC numbers. An editorial in the Journal of Hepatology raised the question ''of whether all liver injuries lead to KC loss". 5 We would, therefore, like to report data on the hepatic immune cell composition in one of the most frequently used animal models in liver cancer studies: diethylnitrosamine (DEN)-induced hepatocellular carcinoma in mice. Although it has been shown before that short-term DEN treatment drives severe liver injury and acute hepatic inflammation, 6 the effects of DEN administration on the composition of myeloid cells are largely unknown. Therefore, we performed a comprehensive flow cytometric analysis upon short-term high-dose DEN treatment to model acute severe liver damage and longterm low-dose DEN treatment to induce hepatocarcinogenesis. The proportion of leukocytes (CD45 + cells) in liver cell suspensions was significantly higher in the DEN-treated animals in the short-term (2.5 ± 0.4-fold in DEN-treated compared to sham-treated livers; p = 0.004), as well as the long-term model (2.10 ± 0.26-fold in DEN-treated compared to sham-treated livers; p = 0.003). The proportion of hepatic macrophages (CD11b + CD11c À NK1.1 À Ly6G À Ly6C lo F4/80 hi) within the leukocyte fraction was significantly decreased in the short-term DEN-treated mice (Fig. 1A). Accordingly, the monocyte/macrophage ratio was highly elevated (19.1 ± 2.0 in DEN-treated mice compared to 2.2 ± 0.4 sham-treated mice; p = 2.39EÀ7). This finding might be interpreted as a KC depletion. However, normalisation of monocyte and macrophage counts to total cell number showed that, while there was indeed an increase in the number of monocytes, no macrophage depletion in the short-term DEN model can be concluded (Fig. 1A). Concordantly, the expression analysis of the C-type lectin domain family 4, member F (Clec4f), which is a specific marker for KCs, revealed no differences between the DEN-and sham-treated animals, neither in the short-term nor in the long-term model (p = 0.62 and p = 0.66, respectively; Fig. 1B). In long-term DEN-treated mice, an increase in both hepatic monocyte and macrophage counts was observed (Fig. 1A). This might be a result of monocytes giving rise to tissue-resident macrophages. Since Clec4f was not elevated in the long-term DEN model, the source and characteristics of the increased proportion of hepatic macrophages, i.e., whether they are derived from infiltrated monocytes and whether they represent a transient state of differentiation towards ...

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