Epigenetic silencing of glutaminase 2 in human liver and colon cancers

Jianbin Zhang,Liting Chen,Ying Zhong,Mingquan Chen,Cheng Wang,Han-Ming Shen,Jianping Cao,Dajing Xia

doi:10.1186/1471-2407-13-601

Abstract

BackgroundGlutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers.MethodsmRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test.ResultsFirst, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest.ConclusionsWe provide evidence showing that epigenetic silencing of Gls2 via promoter hypermethylation is common in human liver and colon cancers and Gls2 appears to be a functional tumor suppressor involved in the liver and colon tumorigenesis.

Highlights

Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism
Demethylation treatment increased Gls2 expression in human liver and colon cancer cells Using CpG island searcher, two typical CGIs were found around Gls2 exon 1 using the following criteria: GC content > 55%, Observed CpG/Expected CpG > 0.65, and length > 500 bp (Figure 2A), suggesting that DNA methylation may occur in the promoter region of Gls2
In order to know whether Gls2 expression is correlated to DNA methylation, both liver and colon cancer cells were treated with a demethylation reagent Aza

Summary

Introduction

Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls has been reported to be downregulated in human hepatocellular carcinomas (HCC). It has been well established that hepatitis virus infection and environmental hepatocarcinogens such as aflatoxins are the main etiological factors [2,3], while the DNA methylation occurs mainly at the cytosine bases of CpG island (CGI) which is found in the promoter region of genes and plays important roles in controlling gene transcription [6,7]. DNA hypermethylation acts as an alternative mechanism of inactivation of tumor suppressor genes, and it is recognized as an important mechanism during tumor initiation and progression [7,8]. In HCC, DNA methylation has been described in many tumor suppressor genes, such as p53, p16INK4a, adenomatous polyposis coli (APC) and Ras association domain-containing protein 1 (RASSF1a) [9,10,11]. Since many tumor suppressor genes are silenced by DNA methylation, DNA methylation has been proposed as a marker to identify novel tumor suppressor genes [10,11,12]

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