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

<p>Abstract</p> <p>Background</p> <p>Hepatoblastoma (HB) is an embryonal liver neoplasm of early childhood with a poor prognosis for patients with distant metastases and vascular invasion. We and others have previously shown that the overexpression of <it>insulin-like growth factor 2 </it>(<it>IGF2</it>), loss of imprinting at the <it>IGF2</it>/<it>H19 </it>locus, and amplification of <it>pleomorphic adenoma gene 1 </it>(<it>PLAG1</it>) are common features in HB, suggesting a critical role of the IGF axis in hepatoblastomagenesis. In this study, we investigated the role of the insulin-like growth factor binding protein 3 (IGFBP3), a known competitor of the IGF axis, in pediatric liver cancers.</p> <p>Results</p> <p>The <it>IGFBP3 </it>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 <it>IGFBP3 </it>promoter region by bisulfite sequencing revealed a high degree of DNA methylation, which is causatively associated with the suppression of <it>IGFBP3 </it>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 <it>IGFBP3 </it>expression. Interestingly, <it>IGFBP3 </it>promoter methylation predominantly occurred in metastatic HB with vascular invasion. Restoring <it>IGFBP3 </it>expression in HB cells resulted in reduced colony formation, migration, and invasion.</p> <p>Conclusion</p> <p>This study provides the first direct evidence that the reactivation of <it>IGFBP3 </it>decreases aggressive properties of pediatric liver cancer cells and that <it>IGFBP3 </it>promoter methylation might be used as an indicator for vessel-invasive tumor growth in HB patients.</p

Downregulation of SFRP1 is a protumorigenic event in hepatoblastoma and correlates with beta-catenin mutations

Background Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are the most common malignant liver tumors in childhood. Both tumor types exhibit genetic and epigenetic alterations in the WNT/beta-catenin signaling pathway, which is a key regulator of liver progenitor cells in embryonic development. The tumors demonstrate a high rate of beta-catenin mutations and gene expression changes of several WNT antagonists. However, the role of the WNT inhibitory factor secreted frizzled-related protein 1 (SFRP1) has not been addressed in pediatric liver cancer so far. Results In our study, we investigated the gene expression level, DNA methylation status and functional relevance of SFRP1 in HB cell lines and in pediatric liver tumor patient samples. SFRP1 was downregulated due to DNA promoter methylation in all tested HB cell lines. Overexpression of SFRP1 in HB cell lines diminished tumor cell proliferation, colony formation and migration potential. In addition, the SFRP1-expressing HB cell lines showed reduced WNT/beta-catenin signaling pathway activity and decreased expression of WNT target genes. To evaluate the utility of SFRP1 as a biomarker in pediatric liver cancer, we determined the gene expression level and DNA methylation status of SFRP1 in 45 pediatric liver tumor patient samples. The correlation analysis of different clinical parameters and tumor characteristics revealed a significant correlation of reduced SFRP1 expression with the presence of mutant beta-catenin. The methylation status of SFRP1 was furthermore associated to a pediatric liver tumor type with HCC-like characteristics, TERT mutations and an older age at diagnosis. Conclusion Altogether, our data demonstrate that the epigenetic suppression of the WNT/beta-catenin antagonist SFRP1 has an important impact on the malignant behavior of HB cells. Although SFRP1 methylation is a common event in HCC-like pediatric liver tumors, its potential as a prognostic or diagnostic biomarker needs to be further investigated