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Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma.

ABSTRACT: Hexokinase 2 (HK2) is a rate-determining enzyme in aerobic glycolysis, a process upregulated in tumor cells. HK2 expression is controlled by various transcription factors and epigenetic alterations and is heterogeneous in hepatocellular carcinomas (HCCs), though the cause of this heterogeneity is not known. DNA methylation in the HK2 promoter CpG island (HK2-CGI) and its surrounding regions (shore and shelf) has not previously been evaluated, but may provide clues about the regulation of HK2 expression. Here, we compared HK2 promoter methylation in HCCs and adjacent non-cancerous liver tissues using a HumanMethylation450 BeadChip array. We found that, while the HK2-CGI N-shore was hypomethylated, thereby enhancing HK2 expression, the HK2-CGI was itself hypermethylated in some HCCs. This hypermethylation suppressed HK2 expression by inhibiting interactions between HIF-1α and a hypoxia response element (HRE) located at -234/-230. HCCs that were HK2negative and had distinct promoter CGI methylation were denoted as having a HK2-CGI methylation phenotype (HK2-CIMP), which was associated with poor clinical outcome. These findings indicate that HK2-CGI N-shore hypomethylation and HK2-CGI hypermethylation affect HK2 expression by influencing the interaction between HIF 1α and HRE. HK2-CGI hypermethylation induces HK2-CIMP and could represent a prognostic biomarker for HCC.

SUBMITTER: Lee HG

PROVIDER: S-EPMC5173097 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma.

Lee Hyun Gyu HG Kim Hyemi H Son Taekwon T Jeong Youngtae Y Kim Seung Up SU Dong Seung Myung SM Park Young Nyun YN Lee Jong Doo JD Lee Jae Myun JM Park Jeon Han JH

Oncotarget 20160701 27

Hexokinase 2 (HK2) is a rate-determining enzyme in aerobic glycolysis, a process upregulated in tumor cells. HK2 expression is controlled by various transcription factors and epigenetic alterations and is heterogeneous in hepatocellular carcinomas (HCCs), though the cause of this heterogeneity is not known. DNA methylation in the HK2 promoter CpG island (HK2-CGI) and its surrounding regions (shore and shelf) has not previously been evaluated, but may provide clues about the regulation of HK2 exp ...[more]

PMID: 27260001

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Project description:AimTo establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC).MethodsThe methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients.ResultsWhile 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues.ConclusionHypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.

Project description:BackgroundmiR-4270 is a regulatory factor has been linked with the progression of various cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma (HCC), and gastric cancer. However, the underlying mechanisms through which miR-4270 modulates HCC development are not fully understood.MethodsmiR-4270 expression levels were analyzed in various HCC cell lines and tissue samples. An online bioinformatics tool was then utilized to predict the miR-4270 target gene. The binding relationship between miR-4270 and its target gene DNMT3A was verified using dual-luciferase reporter and Ago2-RIP assays. Then, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were conducted to investigate the association between DNMT3A and the hepatocyte growth factor activator (HGFAC) promoter region. To assess the methylation level of the HGFAC promoter, methylation-specific PCR (MSP) was employed. Furthermore, rescue analyses were carried out to evaluate the functional relevance of miR-4270 and HGFAC in the modulation of the malignant properties of HCC cells. Finally, HepG2 cells overexpressing miR-4270 were subcutaneously injected into nude mice to estimate the impact of miR-4270 on the xenograft tumor growth of HCC.ResultsA substantial miR-4270 downregulation was revealed in HCC patient samples and cell lines. miR-4270 upregulation suppressed both cell proliferation and invasion while promoting apoptosis. At the molecular level, miR-4270 was found to bind to the 3'untranslated region (3'UTR) of DNMT3A, thereby inhibiting DNMT3A-mediated methylation of the HGFAC promoter. Functional assays indicated that inhibition of miR-4270 stimulated HCC cell growth, an effect counteracted by overexpression of HGFAC. In vivo assays further verified that miR-4270 effectively suppressed the progression of HCC xenograft tumors.ConclusionsmiR-4270 was found to mitigate the malignant characteristics of HCC by inhibiting DNMT3A-mediated methylation of the HGFAC promoter, suggesting a potential therapeutic avenue for the management of HCC.

Project description:BackgroundRecently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.ResultsDifferentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.ConclusionsUsing this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

Dataset Information

Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma.

Publications

Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma.

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