IGFBP3 impedes aggressive growth of pediatric liver cancer and is epigenetically silenced in vascular invasive and metastatic tumors

The IGFBP3 gene was highly expressed in normal pediatric livers but was heavily downregulated in four HB cell lines and the majority of HB primary tumors (26/36). Detailed methylation analysis of CpG sites in the IGFBP3 promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of IGFBP3 in HB cell lines. Consequently, the treatment of HB cell lines with 5-aza-2'-deoxycytidine resulted in DNA demethylation and reactivation of the epigenetically silenced IGFBP3 expression. Interestingly, IGFBP3 promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring IGFBP3 expression in HB cells resulted in reduced colony formation, migration, and invasion.This study provides the first direct evidence that the reactivation of IGFBP3 decreases aggressive properties of pediatric liver cancer cells and that IGFBP3 promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.Hepatoblastoma (HB) represents the most common primary liver tumor in childhood with an incidence of approximately one new case per million children less than 15 years of age [1]. Pathohistologically, HB resembles various stages of the developing liver, showing malignant epithelial cells with fetal and/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 generally responds well to chemotherapy and the prognosis is usually good [2], the outcome of high-risk patients with metastatic tumors or invasion of large hepatic veins is fatal [3,4].The type 1 insulin-like growth factor receptor and its ligands, IGF1 and IGF2, are upregulated in a variety of human cancers [5]. In pediatric tumors, such as rhabdomyosarcoma, nephroblastoma, and HB, the role of the IGF axis is particularly important [6]. We and oth