Intranuclear inclusions in hepatocellular carcinoma contain autophagy‐associated proteins and correlate with prolonged survival

Abstract

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non‐tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane‐bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy‐associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co‐localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy‐associated proteins and proteases within the inclusions contribute to beneficial survival.

Characterization of two types of intranuclear hepatocellular inclusions in NAFLD

“…The lysosome proteases cathepsin B and D together with the presence of heterolysosomes in TEM are suggestive for an active proteolytic activity in type1 NI. This proteolytic activity could also explain the nearby degenerated cell organelles, which we and others have observed in NI 20,21,33,34 . This hypothesis is supported by the identification of autophagy related proteins such as LC3B, p62, ubiquitin and cathepsins in NI.…”

“…Nevertheless, our study shows that there is a significant correlation between the number of NI and disease progression; this is also consistent with the literature. Though NI can be observed both in normal and neoplastic cells 6,32,33 , they are more frequently found in neoplastic cells 20 and are associated with clinical conditions such as diabetes, inflammatory, arteriosclerotic and neoplastic lesions, obesity and NAFLD 8,9,35 . It is documented that NI can be caused by damaging external influences, for example NI induction was observed in mouse livers after colchicine application and in kidney epithelia after chronic lead poisoning and X-ray irradiation 17 .…”

“…Liver samples of each patient were retrieved from the files of the Institute of Pathology, University Hospital of Essen, Germany. In all cases standard processed formalin fixed and paraffin embedded (FFPE) material was stained with HE and immunohistochemical staining was performed according to institutional standards as described before 20 . From each patient an informed consent was received.…”

“…From each paraffin block, additional Sections (3-5 μm) were cut, dewaxed and pretreated. Immunohistochemistry was performed as described previously 20 ; immunostainings of p62, ubiquitin, LC3B, cathepsin B and cathepsin D were carried out on an automated staining device (Dako Autostainer, Dako, Glostrup, Denmark). The antibodies used were: anti-p62 (#sc-28359, Santa Cruz Biotechnology, CA, USA; diluted 1:1,000 for 30 min at RT), anti-cathepsin B (#sc-6490-R, Santa Cruz Biotechnology; diluted 1:500 for 30 min at RT), anti-cathepsin D (#sc-6486, Santa Cruz Biotechnology; diluted 1:6,000 for 60 min at RT), anti-ubiquitin (#Z0458, Dako; diluted 1:1,000 for 30 min at RT) and anti-LC3B (#3868, Cell Signaling Danvers, MA, USA; diluted 1:20 for 60 min at RT).…”

“…In NAFLD, nuclear vacuolation is linked to aging process or senescence but no differentiation is made between NI with or without a limiting membrane 19 . Our recent study of membrane-bounded NI in HCC 20 and in thyroid carcinomas 21 has shown the presence of autophagy-associated proteins and proteases within the inclusions; in HCC the occurrence of inclusions was associated with patients' survival benefit 20 . Jaskolski et al described that autophagy is involved in the pathogenesis of intranuclear vacuolation in meningeomas 22 ; their electron microscopic studies revealed lysosomal bodies and autophagic vacuoles within these NI, suggesting an active macroautophagy process 22 .…”