Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma
Diego F. Calvisi, … , Valentina M. Factor, Snorri S. Thorgeirsson
Diego F. Calvisi, … , Valentina M. Factor, Snorri S. Thorgeirsson
Published September 4, 2007
Citation Information: J Clin Invest. 2007;117(9):2713-2722. https://doi.org/10.1172/JCI31457.
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Research Article Oncology

Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.

Authors

Diego F. Calvisi, Sara Ladu, Alexis Gorden, Miriam Farina, Ju-Seog Lee, Elizabeth A. Conner, Insa Schroeder, Valentina M. Factor, Snorri S. Thorgeirsson

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Figure 1

Analysis of DNA hypomethylation, degree of genomic instability, and CpG hypermethylation in normal livers (NL), nonneoplastic surrounding liver tissues (ST), and HCC with shorter (subclass A) and longer (subclass B) survival rate.

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Analysis of DNA hypomethylation, degree of genomic instability, and CpG ...
(A) As a measure of genome-wide hypomethylation, the incorporation of [3H]dCTP into DNA was assessed after treatment with HpaII endonuclease, cutting only nonmethylated sites. In nontumorous surrounding livers, global DNA hypomethylation was significantly higher than in normal livers. Genomic hypomethylation progressively increased from nonneoplastic surrounding livers to HCC. Liver tumors with poor prognosis displayed the highest levels of DNA hypomethylation. (B) Degree of genomic alterations as detected by RAPD analysis. The level of genomic alterations was significantly higher in subclass A than in subclass B HCC (P = 7.8 × 10–8). (C) Correlation between DNA hypomethylation and genomic alterations. r2 = 0.945; P < 0.001. (D) To determine CpG regional hypermethylation, DNA was pretreated with BssHII endonuclease. Since BssHII cuts only unmethylated sites, the lower [3H]dCTP incorporation indicates fewer unmethylated (cleaved) CpG sites and, therefore, a higher de novo methylation. CpG methylation levels were highest in HCC, particular in tumors of subclass A.