Hepatocyte KLF6 expression affects FXR signalling and the clinical course of primary sclerosing cholangitis

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic cholestasis and inflammation, which promotes cirrhosis and an increased risk of cholangiocellular carcinoma (CCA). The transcription factor Krueppel-like-factor-6 (KLF6) is a mediator of liver regeneration, steatosis, and hepatocellular carcinoma (HCC), but no data are yet available on its potential role in cholestasis. Here, we aimed to identify the impact of hepatic KLF6 expression on cholestatic liver injury and PSC and identify potential effects on farnesoid-X-receptor (FXR) signalling. Methods: Hepatocellular KLF6 expression was quantified by immunohistochemistry (IHC) in liver biopsies of PSC patients and correlated with serum parameters and clinical outcome. Liver injury was analysed in hepatocyte-specific Klf6-knockout mice following bile duct ligation (BDL). Chromatin-immunoprecipitation-assays (ChIP) and KLF6-overexpressing HepG2 cells were used to analyse the interaction of KLF6 and FXR target genes such as NR0B2. Results: Based on IHC, PSC patients could be subdivided into two groups showing either low (80%) hepatocellular KLF6 expression. In patients with high KLF6 expression, we observed a superior survival in Kaplan-Meier analysis. Klf6-knockout mice showed reduced hepatic necrosis following BDL when compared to controls. KLF6 suppressed NR0B2 expression in HepG2 cells mediated through binding of KLF6 to the NR0B2 promoter region. Conclusion: Here, we show an association between KLF6 expression and the clinical course and overall survival in PSC patients. Mechanistically, we identified a direct interaction of KLF6 with the FXR target gene NR0B2

β-catenin in intranuclear inclusions of hepatocellular carcinoma

Aim: β-catenin activation is known to promote liver regeneration and play a role in the pathogenesis of liver cancer. Recently, we detected intranuclear inclusions (NI) in hepatocellular carcinoma (HCC) containing degenerated cell organelles and lysosomal proteins and delimited by a completely closed nuclear membrane. The presence of NI was positively associated with patient survival. The aim of the current study was to investigate a possible association between proteins of the Wnt/β-catenin pathway with NI morphology and survival.Methods: We examined NI in 72 paraffin-embedded specimens of HCC. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed to investigate the content and shape of NI. β-catenin gene (CTNNB1 ) mutations were analyzed by next generation sequencing.Results: We detected the accumulation of β-catenin and glutamine synthetase (a target gene of β-catenin) proteins within NI. Further, we found immunopositivity for the lysine demethylase KDM2A in NI. KDM2A is known to be involved in β-catenin degradation. We detected significant associations between the presence of β-catenin and autophagy-associated proteins in NI. Double-IF revealed co-localization of β-catenin and p62 in the same NI. Kaplan-Meier survival analysis showed that the presence of NI containing KDM2A protein accumulations displayed a significant benefit in overall survival.Conclusion: We detected accumulations of β-catenin and proteins associated with the Wnt/β-catenin pathway partly together with autophagy-associated proteins in the same inclusion. Our finding that KDM2A immunopositivity within NIs was associated with favorable clinical outcomes and suggests a biological significance of NI

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND:Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS:NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS:In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS:NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation