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Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

NOTCH receptor signaling plays a pivotal role in liver homeostasis and hepatocarcinogenesis. However, the role of NOTCH pathway mutations and the NOTCH target gene HES5 in liver tumorigenesis are poorly understood. Here we performed whole-exome sequencing of 54 human HCC specimens and compared the prevalence of NOTCH pathway component mutations with the TCGA-LIHC cohort (N = 364). In addition, we functionally characterized the NOTCH target HES5 and the patient-derived HES5-R31G mutation in vitro and in an orthotopic mouse model applying different oncogenic backgrounds, to dissect the role of HES5 in different tumor subgroups in vivo. We identified nonsynonymous mutations in 14 immediate NOTCH pathway genes affecting 24.1% and 16.8% of HCC patients in the two independent cohorts, respectively. Among these, the HES5-R31G mutation was predicted in silico to have high biological relevance. Functional analyses in cell culture showed that HES5 reduced cell migration and clonogenicity. Further analyses revealed that the patient-derived HES5-R31G mutant protein was non-functional due to loss of DNA binding and greatly reduced nuclear localization. Furthermore, HES5 exhibited a negative feedback loop by directly inhibiting the NOTCH target HES1 and downregulated the pro-proliferative MYC targets ODC1 and LDHA. Interestingly, HES5 inhibited MYC-dependent hepatocarcinogenesis, whereas it promoted AKT-dependent liver tumor formation and stem cell features in a murine model. Thus, NOTCH pathway component mutations are commonly observed in HCC. Furthermore, the NOTCH target gene HES5 has both pro- and anti-tumorigenic functions in liver cancer proposing a driver gene dependency and it promotes tumorigenesis with its interaction partner AKT.

Gallbladder carcinoma (GBC) is a biliary tract cancer with few treatment options and poor prognosis. Radical surgery is the only potentially curative treatment option but most patients diagnosed with GBC are unresectable. Thus, there is a great need for the development of new treatment options including targeted therapy. Here, we aimed at identifying deregulated miRNAs and affected pathways involved in GBC development and progression. We performed global miRNA profiling of 40 GBC and 8 normal gallbladder tissues and identified large differences with 30% of miRNAs being differentially expressed (false discovery rate: FDR

UNLABELLED Several chronic inflammatory liver diseases, e.g., chronic hepatitis B or C viral infection and steatohepatitis, have been shown to predispose to the development of hepatocellular carcinoma (HCC). In patients with chronic liver disease, interleukin-6 (IL-6) serum levels are elevated and increase even more when HCC develops. However, the impact and regulatory mechanisms of IL-6 signaling during hepatocarcinogenesis are still poorly defined. Here, we show that gene expression profiles of patients with chromosome 8p loss correlate with increased IL-6 signaling. In addition, the chromosome 8p tumor suppressor genes Src homology 2 domain containing 4A (SH2D4A) and Sorbin and Src homology 3 domain containing 3 (SORBS3) together exerted greater inhibition of cell growth and clonogenicity compared to a single gene. Overexpression of SH2D4A and SORBS3 in HCC cells led to decreased IL-6 target gene expression and reduced signal transducer and activator of transcription 3 (STAT3) signaling. In situ and in vitro coimmunoprecipitation assays revealed that SH2D4A directly interacts with STAT3, thereby retaining STAT3 in the cytoplasm and inhibiting STAT3 transcriptional activity. On the other hand, SORBS3 coactivated estrogen receptor α signaling, leading indirectly to repression of STAT3 signaling. In human HCC tissues, SH2D4A was positively associated with infiltrating regulatory and cytotoxic T-cell populations, suggesting distinct immunophenotypes in HCC subgroups with chromosome 8p loss. Thus, the genetically linked tumor suppressors SH2D4A and SORBS3 functionally cooperate to inhibit STAT3 signaling in HCC. CONCLUSION The chromosome 8p tumor suppressor genes SORBS3 and SH2D4A are physically and functionally linked and provide a molecular mechanism of inhibiting STAT3-mediated IL-6 signaling in HCC cells. (Hepatology 2016;64:828-842).

The NOTCH pathway is an evolutionary conserved signaling pathway that is known to play a pivotal role in physiological liver development and regeneration. Thereby, the NOTCH pathway controls cell fate decisions of bipotential liver progenitor cells promoting a biliary lineage rather than hepatic differentiation. Furthermore, aberrant NOTCH signaling is also a potential driver of liver inflammation, formation and progression of hepatocellular (HCC) and intrahepatic cholangiocarcinoma (iCCA). Previous studies mainly focused on the expression levels of the NOTCH receptors (NOTCH1-4) and their ligands (DLL1/3/4, JAG1/2). However, little is known regarding mutational effects within components of the Notch pathway and their relevance in liver cancer. In a whole exome sequencing approach of 54 human HCC samples, we discovered 19 amino acid-altering mutations in 15 different NOTCH pathway genes which were confirmed by Sanger sequencing to be somatic. In summary, these 15 mutations affected 25.9% of patients (14 out of 54 patients) and presented at least one mutation in one of the included NOTCH pathway components (NOTCH1/3/4, DLL1, DTX1/3/4, HES5, DVL2, LFNG, NUMB, NCSTN, DMXL2, GXYLT2). Consistently, 30% of HCC patients have been reported to contain a tumor-associated hyper-activated NOTCH pathway. Using multiple online tools to predict the effect of the mutations on protein function and tumor relevance, we observed that HES5-Arg31Gly mutation ranked highest. In order to test, whether HES5 is activated by NOTCH in HCC cells, we generated cell lines with inducible expression of NOTCH1 or NOTCH3 intracellular domain and found that the transcription factor HES5 but not HES1 is strongly activated in several HCC cell lines. Furthermore, induction of HES5 in an inducible Hep3B cell line resulted in increased expression of stem cell markers and epithelial-mesenchymal transition markers. In contrary, CCND1, HNF4alpha and HES1 were downregulated. Interestingly, HES5-Arg31Gly failed to modulate the expression of the here tested target genes. The loss of function of HES5-Arg31Gly may be partially explained by lower nuclear translocation of HES5-Arg31Gly protein. In vivo, mouse models using hydrodynamic transduction of transposons expressing MYC and HES5 indicated a tumor suppressive role of HES5, whereas, in AKT-driven liver tumorigenesis, HES5 exhibited oncogenic potential. Consistent with our in vitro experiments, AKT/HES5-driven murine tumors exhibited partial downregulation of HNF4alpha and upregulation of the cholangiocyte marker cytokeratin suggesting a transdifferentiation towards a cholangiocytic phenotype. Thus, we identified somatic mutations in NOTCH pathway genes in 25.9% of HCC patients. Our functional in vitro and in vivo analyses suggest that HES5 has oncogenic or tumor suppressive properties depending on the underlying oncogene or tumor subtype, respectively. Citation Format: Sarah Luiken, Matthias Bieg, Angelika Fraas, Raisatun Sugiyanto, Benjamin Goeppert, Stephan Singer, Stefan Pusch, Arianeb Mehrabi, Thomas Longerich, Peter Schirmacher, Stephanie K. Roessler. NOTCH/HES5 signaling exhibits distinct pro- and anti-tumorigenic roles in liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3487.