CLINICAL STUDIES Aberrant Notch3 and Notch4 expression in human hepatocellular carcinoma Laura Gramantieri 1,2Ã , Catia Giovannini 1,2Ã , Arianna Lanzi 1,2 , Pasquale Chieco 1 , Matteo Ravaioli 3 , Annamaria Venturi 1,2 , Gian Luca Grazi 3 and Luigi Bolondi 1,2 1 Center for Applied Biomedical Research (CRBA), University of Bologna and St Orsola-Malpighi University Hospital, Bologna, Italy 2 Department of Internal Medicine and Gastroenterology, University of Bologna and St Orsola-Malpighi University Hospital, Bologna, Italy 3 Department of Surgery, University of Bologna and St Orsola-Malpighi University Hospital, Bologna, Italy Keywords HCC – HES1 – HES6 – Notch3 – Notch4 Correspondence Laura Gramantieri, MD, PhD, Divisione di Medicina Interna, Policlinico St Orsola-Malpighi, Via Albertoni, 15 40138 Bologna, Italy Tel: 139 051 6362260 Fax: 139 051 6362725 e-mail: gramantieri@aosp.bo.it Received 21 December 2006 accepted 17 May 2007 DOI:10.1111/j.1478-3231.2007.01544.x Abstract Background: Notch signalling is altered in several solid tumours and it plays a role in growth inhibition and apoptosis of hepatocellular carcinoma (HCC)-derived cell lines, bile duct development and hepatocyte regeneration. Aims: This study aims to analyse the expression of Notch3, Notch4 and HES1 and HES6 as Notch-target genes in HCC, matched non-neoplastic tissue and HEPG2 cells. Results: Notch3 and Notch4 are not expressed in normal liver and in chronic hepatitis surrounding HCC. Cirrhotic tissue stains negative for Notch3, while Notch4 is expressed by hepatocytes at the edge of regenerative nodules and in cell planes adjacent to fibrous septa. HCC tissue displays Notch3 and Notch4 abnormal accumulation, respectively, in 78% and 68% of the cases. The endothe- lium of hepatic veins with neoplastic permeation is frequently Notch4 positive. An upregulation of Notch3 mRNA was found in 95% of HCCs vs cirrhosis (P = 0.0001), while Notch4 mRNA was downregulated in 80% of HCCs. HES6 mRNA expression was higher in HCC tissue when compared with cirrhosis (P = 0.007), paralleling Notch3 mRNA expression. The HEPG2 cell line displays high Notch3 and low Notch4 protein and mRNA levels. Conclusions: These descriptive findings suggest an aberrant expression of Notch3 and Notch4 in HCC and allow the hypothesis of an activation of Notch signalling by Notch3. The Notch gene family encodes large transmembrane receptors holding an extracellular domain with many EGF-like repeats, followed by three cystein-rich Notch/lin12 repeats and a cytoplasmic domain that conveys the signal to the nucleus. Four Notch recep- tors (Notch1–4) and five ligands (Delta-like1, 3 and 4 and Jagged1 and 2) have been identified in humans. Notch signalling is initiated by a receptor–ligand interaction between two neighbouring cells, which triggers a two-step proteolytic cleavage that releases the cytoplasmic portion of Notch that translocates to the nucleus and modulates the transcription of target genes. To date, the Hairy/Enhancer of Split (HES) and HES-related repressor protein (HERP) families of transcriptional repressors are the only known major targets of Notch signalling (1–3). The Notch pathway interacts with several other signal transduction pathways, and its activation can lead to different outcomes that are cell type and context specific, ranging from control of proliferation to apoptosis, differentiation, maintenance of stemness and cell fate decision (4). The involvement of the Notch signalling pathway in neoplastic transformation has been recognized in several human neoplasms and its role appears to be tumour-type dependent (5–10). Even if most studies have been focused on Notch1, Notch3 and Notch4 may also be involved in malignant transformation. Aberrant Notch3 expression was reported in T-cell leukaemia (11) and Notch4 has been implicated in the proliferation and differentiation of haematopoietic precursors (12) and in mouse mam- mary gland tumours, where Notch4 is a common integration site for the mammary tumour virus, which leads to the ectopic expression of the Notch4 intracellular domain (13). Other functions attributed to Notch signalling are those related to vascular development and homeostasis and lateral inhibition (14). Ã Contributed equally to this work. Liver International (2007) c  2007 The Authors. Journal compilation c  2007 Blackwell Munksgaard 997 Liver International ISSN 1478-3223