Page 58 - Read Online

Page 2 of 14 Schwertheim et al. Hepatoma Res 2020;6:41 I http://dx.doi.org/10.20517/2394-5079.2020.23

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.

Keywords: Wnt/β-catenin pathway, KDM2A, intranuclear inclusions, hepatocellular carcinoma

INTRODUCTION
The Wnt/β-catenin signaling pathway is regarded as playing the most important role in hepatocellular
[1-4]
[4]
regeneration . Spee et al. reported that the Wnt/β-catenin signaling pathway is active in the ductular
[4,5]
[1]
reaction of acute liver failure patients . Apte et al. demonstrated a significant correlation between
nuclear/cytoplasmic-β-catenin staining and hepatocellular regeneration. Recently, we published a detailed
study on intranuclear inclusions (NI) in hepatocellular carcinoma (HCC). In general, NI are considered
to be a morphological feature, without any notable function or influence on disease. On the contrary, our
earlier studies revealed that these NI are surrounded by a completely closed nuclear membrane and contain
degenerated organelles, autophagy-associated proteins and lysosomes, suggesting a biological function of NI.
[6]
The presence of NI was also significantly correlated with survival in HCC . However, the factors that play
a role in the occurrence of NI are mostly still unknown. Thus, we questioned whether proteins of the Wnt/
β-catenin pathway are involved in the functioning of NI.

We performed immnuohistochemical analyses to investigate the presence of β-catenin, glutamine synthetase
[7]
and KDM2A in NI. Glutamine synthetase, an enzyme involved in the removal of ammonia in the liver ,
[8]
[9]
[8]
nitrogen balance and pH regulation is also a target of the Wnt/b-catenin pathway in the liver . Long et al.
showed increased levels of expression of glutamine synthetase in HCC and in liver tissue with cirrhosis and
[10]
chronic hepatitis B. KDM2A (FBXL11) is known to demethylate histone H3K36 , which contains an F-box,
a JmjC domain, a CxxC zinc finger, a PHD domain, and three leucine-rich repeat elements [10-13] . KDM2A
is involved in various signaling pathways including NF-κB signaling, p53 activity and WNT/β-catenin
[14]
pathway; demethylation of β-catenin with consequent degradation has been reported . Thus, to investigate
a possible correlation of KDM2A expression levels on survival in HCC patients, we performed Kaplan-Meier
survival studies. Further, we examined if positive immunoreactivity for β-catenin within NI is associated
simultaneously with the presence of the autophagy-associated proteins p62/sequestosome1, ubiquitin, LC3B,
cathepsin B and cathepsin D in NI.

The goal of the current study was to investigate whether proteins of the Wnt/β-catenin pathway are
associated with NI morphology and disease survival.

METHODS
Patients
Seventy two patients with HCC diagnosed at the Institute of Pathology, University Hospital of Essen,
Germany between 1999 and 2005 were included in this study. Formalin-fixed and paraffin-embedded (FFPE)
material from untreated patients were provided in all cases, prepared according to institutional standards
[15]
[6]
and stained with H&E as described before . The current WHO criteria were used for the diagnosis of all
tumors and the classification of tumors was based on the TNM system (8th edition). Table 1 provides an
overview of patient data and tumor characteristics. In 70 HCC cases, complete clinical records and follow-up
data were available.

Informed consent was obtained from each patient. The study complied with the Helsinki Declaration of 1975
and the Ethics Committee (Institutional Review Board) of the University Hospital Essen (reference number:
16-6917-BO).