Hepatoblastomas exhibit markedNNMTdownregulation driven by promoter DNA hypermethylation

Abstract

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Epigenetics in hepatoblastoma

“…Consistently, overexpression of SFRP1 hampers the Wnt/β-catenin pathway in human HB cell lines, resulting in inhibition of tumor cell growth, colony formation, and migration, whereas SFRP1 knockdown stimulates tumor cell growth [95,96] . Moreover, hypermethylation of the promoters of G protein-coupled receptor 180 (GPR180), macrophae stimulating 1 receptor (MST1R), OCIA domain containing 2 (OCIAD2), and poly(ADP-ribose) polymerase family member 6 (PARP6) genes correlates with metastatic HB tumors and poor outcome [97] . Furthermore, nicotinamide N-methyltransferase (NNMT), a highly expressed gene in hepatocytes involved in the N-methylation of pyridine-containing compounds [98,99] , is hypermethylated and downregulated in HB, in association with late HB diagnosis [98] .…”

Mecanismos epigenéticos em hepatoblastomas: análise do transcriptoma e sua regulação por metilação de DNA

“…Changes in HBL metabolism are still poorly described and understood 167,168 . We previously showed the occurrence of lipid reduction and nicotinamide metabolism changes in HBLs 142 , and this RNASeq analysis reinforces and expands these observations. Genes with changes in expression in HBLs are widely distributed on chromosomes (Figure 1B), but the 14q32 region showed a cluster of upregulated genes.…”

“…On the other hand, all the negatively regulated biological processes in HBL were associated with metabolism and oxidation. Through non-targeted metabolomic analysis, our group recently detected reduced lipid content in HBLs 142 . Wang et al demonstrated metabolic changes in HBLs associated with negative regulation of SLC10A1 and linked its effect to disturbances in the control of cell proliferation 168 , a gene also downregulated in our data.…”

“…Nesse cenário, o gene NNMT era uma proteína interessante a ser estudada, uma vez que é central para o metabolismo de hepatócitos e adipócitos. Havíamos identificado hipermetilação no promotor deste gene nos tumores e, com o aprofundamento do estudo, verificamos que ocorre redução de expressão e consequente diminuição no nível proteíco 142 .…”

“…Recently, we demonstrated NNMT downregulation 142 , which is an enzyme responsible for the nicotinamide N-methylation process and associated with the regulation of metabolic pathways in hepatocytes 121 . Metabolomics analysis detected a reduction in the lipid content of tumors, possibly associated with the NNMT downregulation and suggesting a connection with worse prognosis 142 .…”