The online version of this article (doi:10.1186/1476-4598-11-9) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
IR carried out the processing of clinical samples, expression measurements, migration assays, 5-Aza-dC treatments, and the methylation-specific PCR, and she participated in the analysis and interpretation of the data. ME performed the Western blot and apoptosis assays. SJ conducted the bisulfite-sequencing study and interpreted the methylation data. JL performed the invasion and migration assays. BH collected the clinical samples and carried out the statistical analyses of the clinical data. JMH performed the pathological diagnosis and staging of tumor specimens. AV participated in the design of the study and helped in the analysis and interpretation of the data. DvS participated in the design of the study and helped to draft the manuscript. RK designed the study, participated in the analysis and interpretation of data, and drafted the manuscript. All of the authors read and approved the final version of this manuscript.
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 insulin-like growth factor 2 (IGF2), loss of imprinting at the IGF2/H19 locus, and amplification of pleomorphic adenoma gene 1 (PLAG1) 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.
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.
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Fuchs J, Rydzynski J, Von Schweinitz D, Bode U, Hecker H, Weinel P, Burger D, Harms D, Erttmann R, Oldhafer K, Mildenberger H: Pretreatment prognostic factors and treatment results in children with hepatoblastoma: a report from the German Cooperative Pediatric Liver Tumor Study HB 94. Cancer. 2002, 95: 172-182. 10.1002/cncr.10632 CrossRefPubMed
Hartmann W, Waha A, Koch A, Goodyer CG, Albrecht S, von Schweinitz D, Pietsch T: p57(KIP2) is not mutated in hepatoblastoma but shows increased transcriptional activity in a comparative analysis of the three imprinted genes p57(KIP2), IGF2, and H19. Am J Pathol. 2000, 157: 1393-1403. 10.1016/S0002-9440(10)64652-4 PubMedCentralCrossRefPubMed
Li X, Adam G, Cui H, Sandstedt B, Ohlsson R, Ekstrom TJ: Expression, promoter usage and parental imprinting status of insulin-like growth factor II (IGF2) in human hepatoblastoma: uncoupling of IGF2 and H19 imprinting. Oncogene. 1995, 11: 221-229. PubMed
Honda S, Arai Y, Haruta M, Sasaki F, Ohira M, Yamaoka H, Horie H, Nakagawara A, Hiyama E, Todo S, Kaneko Y: Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma. Br J Cancer. 2008, 99: 1891-1899. 10.1038/sj.bjc.6604754 PubMedCentralCrossRefPubMed
Modric T, Silha JV, Shi Z, Gui Y, Suwanichkul A, Durham SK, Powell DR, Murphy LJ: Phenotypic manifestations of insulin-like growth factor-binding protein-3 overexpression in transgenic mice. Endocrinology. 2001, 142: 1958-1967. 10.1210/en.142.5.1958 PubMed
Rajah R, Valentinis B, Cohen P: Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGF-independent mechanism. J Biol Chem. 1997, 272: 12181-12188. 10.1074/jbc.272.18.12181 CrossRefPubMed
Williams AC, Collard TJ, Perks CM, Newcomb P, Moorghen M, Holly JM, Paraskeva C: Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells. Cancer Res. 2000, 60: 22-27. PubMed
Zou T, Fleisher AS, Kong D, Yin J, Souza RF, Wang S, Smolinski KN, Abraham JM, Meltzer SJ: Sequence alterations of insulin-like growth factor binding protein 3 in neoplastic and normal gastrointestinal tissues. Cancer Res. 1998, 58: 4802-4804. PubMed
Hanafusa T, Yumoto Y, Nouso K, Nakatsukasa H, Onishi T, Fujikawa T, Taniyama M, Nakamura S, Uemura M, Takuma Y: Reduced expression of insulin-like growth factor binding protein-3 and its promoter hypermethylation in human hepatocellular carcinoma. Cancer Lett. 2002, 176: 149-158. 10.1016/S0304-3835(01)00736-4 CrossRefPubMed
Gray SG, Kytola S, Lui WO, Larsson C, Ekstrom TJ: Modulating IGFBP-3 expression by trichostatin A: potential therapeutic role in the treatment of hepatocellular carcinoma. Int J Mol Med. 2000, 5: 33-41. PubMed
Luo SM, Tan WM, Deng WX, Zhuang SM, Luo JW: Expression of albumin, IGF-1, IGFBP-3 in tumor tissues and adjacent non-tumor tissues of hepatocellular carcinoma patients with cirrhosis. World J Gastroenterol. 2005, 11: 4272-4276. PubMed
de Ibanez Caceres I, Dulaimi E, Hoffman AM, Al-Saleem T, Uzzo RG, Cairns P: Identification of novel target genes by an epigenetic reactivation screen of renal cancer. Cancer Res. 2006, 66: 5021-5028. 10.1158/0008-5472.CAN-05-3365 CrossRef
Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG, Esteller M: A systematic profile of DNA methylation in human cancer cell lines. Cancer Res. 2003, 63: 1114-1121. PubMed
Chang YS, Wang L, Liu D, Mao L, Hong WK, Khuri FR, Lee HY: Correlation between insulin-like growth factor-binding protein-3 promoter methylation and prognosis of patients with stage I non-small cell lung cancer. Clin Cancer Res. 2002, 8: 3669-3675. PubMed
Calvisi DF, Ladu S, Gorden A, Farina M, Lee JS, Conner EA, Schroeder I, Factor VM, Thorgeirsson SS: Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest. 2007, 117: 2713-2722. 10.1172/JCI31457 PubMedCentralCrossRefPubMed
Aishima S, Basaki Y, Oda Y, Kuroda Y, Nishihara Y, Taguchi K, Taketomi A, Maehara Y, Hosoi F, Maruyama Y: High expression of insulin-like growth factor binding protein-3 is correlated with lower portal invasion and better prognosis in human hepatocellular carcinoma. Cancer Sci. 2006, 97: 1182-1190. 10.1111/j.1349-7006.2006.00322.x CrossRefPubMed
Kappler R, von Schweinitz D: Molecular aspects of hepatoblastoma. Pediatric Liver Tumors. Edited by: Zimmermann A, Perilongo G. 2011, 27-42. Heidelberg: Springer, CrossRef
Pietsch T, Fonatsch C, Albrecht S, Maschek H, Wolf HK, von Schweinitz D: Characterization of the continuous cell line HepT1 derived from a human hepatoblastoma. Lab Invest. 1996, 74: 809-818. PubMed
Specht K, Kremer M, Muller U, Dirnhofer S, Rosemann M, Hofler H, Quintanilla-Martinez L, Fend F: Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections. Clin Cancer Res. 2002, 8: 2902-2911. PubMed
Eichenmuller M, von Schweinitz D, Kappler R: Betulinic acid treatment promotes apoptosis in hepatoblastoma cells. Int J Oncol. 2009, 35: 873-879. PubMed
- IGFBP3 impedes aggressive growth of pediatric liver cancer and is epigenetically silenced in vascular invasive and metastatic tumors
Dietrich von Schweinitz
- BioMed Central
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