Recent evidence has indicated that Hedgehog (Hh) signaling significantly contributes to liver development and regeneration and that activation of the pathway may contribute to growth of hepatocellular carcinoma (HCC) in adults. However, the role of Hh signaling in pediatric liver tumors remains to be elucidated. In this study, we show that Hh signaling is activated in hepatoblastoma (HB), the most common liver tumor in childhood, with most occurrences before the age of 3 years. The Hh target genes glioma-associated oncogene homolog 1 (GLI1) and Patched (PTCH1) showed increased transcript levels in 65% and 30% of HB samples, respectively, compared with normal liver tissues. Most interestingly, the gene encoding the hedgehog interacting protein ( HHIP) is transcriptionally silenced by cytosinephospho-guanosine (CpG) island promoter hypermethylation in 26% of HB cases and treatment with the DNA-demethylating agent 5-aza-2-deoxycytidine partially restored HHIP expression. Blocking Hh signaling with the antagonist cyclopamine had a strong inhibitory effect on cell proliferation of HB cell lines with an activated pathway. We further demonstrate that this decrease in cell viability is caused by a massive induction of apoptosis, as shown by morphological changes and phosphatidylserine membrane asymmetry. In cyclopamine-exposed HB cells, caspase 3 and poly(adenosine diphosphate-ribose) polymerase proteins were specifically activated by their proteolytic cleavage. Conclusion: This study demonstrates, for the first time, the frequent occurrence of GLI1 and PTCH1 overexpression and HHIP promoter methylation in early childhood HB, thus indicating a key role for Hh signaling activation in the malignant transformation of embryonal liver cells. (HEPATOLOGY 2009;49:482-490.)
H epatoblastoma (HB) represents the most common malignant liver tumor in children with a median age at presentation of 6 to 36 months. 1 The incidence in Western countries is estimated to be approximately one HB case per million children, 2 with an increasing trend corresponding to an improved survival of very-low-birth-weight babies. 3 Pathohistologically, HB resembles various stages of the developing liver, showing malignant epithelial cells with fetal or embryonal hepatic differentiation and foci of primitive blastemal cells. The mixed HB subtype also contains interspersed mesenchymal elements such as immature fibrous tissue, spindle cells, and osteoid. 1 Although HB treatment has dramatically improved during the past 20 years by combining chemotherapy regimens with surgery, 4 the fatal outcome of high-risk patients with advanced or recurrent HB makes new treatment strategies essential. 5,6 Accumulated experimental evidence indicates that Hedgehog (Hh) signaling is implicated in embryonic development of the liver and its regeneration in the adult. It