American Association for Cancer Research
00085472can103823-sup-suppl__fig__s4_pdf_1468k.pdf (1.43 MB)

Supplementary Figure 4 from Definition of the Landscape of Promoter DNA Hypomethylation in Liver Cancer

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journal contribution
posted on 2023-03-30, 20:53 authored by Barbara Stefanska, Jian Huang, Bishnu Bhattacharyya, Matthew Suderman, Michael Hallett, Ze-Guang Han, Moshe Szyf

PDF file - 1468K, Methylation and expression of MMP2, NUPR1, S100A5, and PLAU in NorHep treated with 5-azaCdR at 1.0 �M concentration. (A) Global methylation level estimated using luminometric methylation assay (LUMA) in NorHep treated with 5-azaCdR for 5 or 20 days. LUMA assay and treatment with 5-azaCdR were performed as described in the Supplementary Methods. (B) Fold change in relative gene expression as measured by QPCR in cells treated with 5-azaCdR for 5 days except for NUPR1 where an increase in expression was observed after 20 days of treatment compared to the control cells cultured in the absence of the drug. (C) State of methylation as determined by pyrosequencing in the promoter region of MMP2, NUPR1, S100A5, and PLAU in NorHep treated with 5-azaCdR for 5 days or 20 days (NUPR1). Methylation level was estimated in the same CpGs as in the experiment with HepG2 cells after MBD2 depletion. All results represent mean � S.D. of two or three independent experiments, measured in triplicate ***P < 0.001, **P <0.01, *P <0.05. (D) Correlation between the extent of promoter demethylation and gene induction for 230 genes epigenetically induced in HCC patients. Each point in the scatter plot represents a differential methylation and differential expression for every gene in each HCC patient. For both differential methylation and differential expression, we used the log-fold change of the microarray probe that the most significantly differentiated between cancer and normal for the given gene. The correlation is negative with Pearson's correlation coefficient -0.1 (P <0.0019).



We use hepatic cellular carcinoma (HCC), one of the most common human cancers, as a model to delineate the landscape of promoter hypomethylation in cancer. Using a combination of methylated DNA immunoprecipitation and hybridization with comprehensive promoter arrays, we have identified approximately 3,700 promoters that are hypomethylated in tumor samples. The hypomethylated promoters appeared in clusters across the genome suggesting that a high-level organization underlies the epigenomic changes in cancer. In normal liver, most hypomethylated promoters showed an intermediate level of methylation and expression, however, high-CpG dense promoters showed the most profound increase in gene expression. The demethylated genes are mainly involved in cell growth, cell adhesion and communication, signal transduction, mobility, and invasion; functions that are essential for cancer progression and metastasis. The DNA methylation inhibitor, 5-aza-2′-deoxycytidine, activated several of the genes that are demethylated and induced in tumors, supporting a causal role for demethylation in activation of these genes. Previous studies suggested that MBD2 was involved in demethylation of specific human breast and prostate cancer genes. Whereas MBD2 depletion in normal liver cells had little or no effect, we found that its depletion in human HCC and adenocarcinoma cells resulted in suppression of cell growth, anchorage-independent growth and invasiveness as well as an increase in promoter methylation and silencing of several of the genes that are hypomethylated in tumors. Taken together, the findings define the potential functional role of hypomethylation in cancer. Cancer Res; 71(17); 5891–903. ©2011 AACR.

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