TET Upregulation Leads to 5-Hydroxymethylation Enrichment in Hepatoblastoma

Abstract

Hepatoblastoma is an embryonal liver tumor carrying few genetic alterations. We previously disclosed in hepatoblastomas a genome-wide methylation dysfunction, characterized by hypermethylation at specific CpG islands, in addition to a low-level hypomethylation pattern in non-repetitive intergenic sequences, in comparison to non-tumoral liver tissues, shedding light into a crucial role for epigenetic dysregulation in this type of cancer. To explore the underlying mechanisms possibly related to aberrant epigenetic modifications, we evaluated the expression profile of a set of genes engaged in the epigenetic machinery related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L, UHRF1, TET1, TET2, and TET3), as well as the 5-hydroxymethylcytosine (5hmC) global level. We observed in hepatoblastomas a general disrupted expression of these genes from the epigenetic machinery, mainly UHRF1, TET1, and TET2 upregulation, in association with an enrichment of 5hmC content. Our findings support a model of active demethylation by TETs in hepatoblastoma, probably during early stages of liver development, which in combination with UHRF1 overexpression would lead to DNA hypomethylation and an increase in overall 5hmC content. Furthermore, our data suggest that decreased 5hmC content might be associated with poor survival rate, highlighting a pivotal role of epigenetics in hepatoblastoma development and progression.

Keywords: DNA hypomethylation; active demethylation; epigenetics; hepatoblastoma; hydroxymethylation.

FIGURE 1

Expression analysis of DNA methylation genes in hepatoblastomas. DNMT1, DNMT3A and UHRF1 upregulation in 19 tumor samples compared with eight non-tumoral liver samples as evidenced by RT-qPCR. Control Liver: non-tumoral liver tissue samples; HB: hepatoblastoma samples. Endogenous gene: 18S rRNA. The Kruskal Wallis post hoc Dunn test with Bonferroni correction was used for statistical analysis. ^∗p-value < 0.05, ^∗∗p-value < 0.01.

FIGURE 2

Analysis of DNA hydroxymethylation in hepatoblastomas. (A) Expression analysis of DNA hydroxymethylation genes in hepatoblastomas. Upregulation of three genes of the TET family in 19 tumor samples compared with eight non-tumoral liver samples as evidenced by RT-qPCR. Control Liver: non-tumoral liver tissue samples; HB: hepatoblastoma samples. Endogenous gene: 18S rRNA. The Kruskal Wallis post hoc Dunn test with Bonferroni correction was used for statistical analysis. ^∗p-value < 0.05; ^∗∗p-value < 0.01. (B) 5hmC global level enrichment in hepatoblastoma samples. Violin plots showing the overall level of 5hmC in percentage in hepatoblastomas samples (red) and non-tumoral liver tissues (blue). Hepatoblastoma samples mean: 0.33%, SD: 0.15; non-tumoral liver tissue samples mean: 0.13%; SD: 0.07. The t-test was used for the statistical analysis. p-value: 4.2e-05.

FIGURE 3

Decreased overall survival is associated with lower levels of 5hmC. This figure shows the 5hmC global content of 18 tumor samples in the columns and their respective clinical data below. Tumor samples with overall survival ≤ 5 years present reduced 5hmC content, below the mean of the tumor group (0.33% - represented by the dashed line) and close to the level of non-tumoral hepatic tissues (CL). The Fisher’s test was used for the statistical analysis. p-value 0.02. CL: non-tumoral liver tissue samples; T: hepatoblastoma tumor samples. ^∗According to the CHIC criteria (Meyers et al., 2017).