Epigenetic silencing of glutaminase 2 in human liver and colon cancers

BMC Cancer

Jianbin Zhang²  Cheng Wang⁴  Mingquan Chen³  Jianping Cao¹  Ying Zhong⁴  Liting Chen⁴  Han-Ming Shen²  Dajing Xia⁴ 

[1] School of Public Health, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China

[2] Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore

[3] Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai 200040, China

[4] Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou 310058, China

关键词: Colon cancer;    Hepatocellular carcinoma;    Methylation;    Gls2;   

Others  :  859191
DOI  :  10.1186/1471-2407-13-601

 received in 2013-07-15, accepted in 2013-12-06,  发布年份 2013

【 摘 要 】

Background

Glutaminase 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.

Methods

mRNA 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.

Results

First, 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.

Conclusions

We 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.

【 授权许可】

   
2013 Zhang et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Yang JD, Roberts LR: Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol 2010, 7(8):448-458.
• [2]Liu Y, Wu F: Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect 2010, 118(6):818-824.
• [3]El-Serag HB: Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 2012, 142(6):1264-1273. e1261
• [4]Jones PA, Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002, 3(6):415-428.
• [5]Nishida N, Goel A: Genetic and epigenetic signatures in human hepatocellular carcinoma: a systematic review. Curr Genomics 2011, 12(2):130-137.
• [6]Jones PA: Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Gene 2012, 13(7):484-492.
• [7]Bergman Y, Cedar H: DNA methylation dynamics in health and disease. Nat Struct Mol Biol 2013, 20(3):274-281.
• [8]Akhavan-Niaki H, Samadani AA: DNA methylation and cancer development: molecular mechanism. Cell Biochem Biophys 2013, 67(2):501-513.
• [9]Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH: Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol 2003, 163(4):1371-1378.
• [10]Herath NI, Leggett BA, MacDonald GA: Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroen Hepatol 2006, 21(1 Pt 1):15-21.
• [11]Pogribny IP, Rusyn I: Role of epigenetic aberrations in the development and progression of human hepatocellular carcinoma. Cancer Lett 2014, 342(2):223-230.
• [12]Baylin SB, Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev Cancer 2006, 6(2):107-116.
• [13]Hu W, Zhang C, Wu R, Sun Y, Levine A, Feng Z: Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function. Proc Natl Acad Sci USA 2010, 107(16):7455-7460.
• [14]Suzuki S, Tanaka T, Poyurovsky MV, Nagano H, Mayama T, Ohkubo S, Lokshin M, Hosokawa H, Nakayama T, Suzuki Y, et al.: Phosphate-activated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive oxygen species. Proc Natl Acad Sci USA 2010, 107(16):7461-7466.
• [15]Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Kravchenko JE, Chumakov PM: The antioxidant function of the p53 tumor suppressor. Nat Med 2005, 11(12):1306-1313.
• [16]D’Autreaux B, Toledano MB: ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 2007, 8(10):813-824.
• [17]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 2001, 25(4):402-408.
• [18]Jones PA, Baylin SB: The epigenetics of cancer. Cell 2007, 128:683-692.
• [19]Nishida N, Kudo M, Nagasaka T, Ikai I, Goel A: Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma. Hepatology 2012, 56(3):994-1003.
• [20]Xie B, Zhou J, Yuan L, Ren F, Liu DC, Li Q, Shu G: Epigenetic silencing of Klotho expression correlates with poor prognosis of human hepatocellular carcinoma. Hum Pathol 2013, 44(5):795-801.
• [21]Riviere G, Lienhard D, Andrieu T, Vieau D, Frey BM, Frey FJ: Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation. Epigenetics: official journal of the DNA Methylation Society 2011, 6(4):478-489.
• [22]Taylor WR, Stark GR: Regulation of the G2/M transition by p53. Oncogene 2001, 20(15):1803-1815.
• [23]Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998, 282(5393):1497-1501.
• [24]Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Vogelstein B: 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell 1997, 1(1):3-11.
• [25]Shaw P, Bovey R, Tardy S, Sahli R, Sordat B, Costa J: Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA 1992, 89(10):4495-4499.
• [26]Clarke AR, Gledhill S, Hooper ML, Bird CC, Wyllie AH: p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation. Oncogene 1994, 9(6):1767-1773.