Early B-cell factor 3 (EBF3) is a novel tumor suppressor gene with promoter hypermethylation in pediatric acute myeloid leukemia

Journal of Experimental & Clinical Cancer Research

Jian Pan³  Wen-Li Zhao³  Feng Xing³  Jian Wang³  Jian Ni⁴  Xue-Ming Zhu³  Lin Liu³  Mei-Fang Jin³  Yi Wu³  Hui-Ting Zhou³  Yun-Yun Xu³  Yi-Ping Li³  He Zhao³  Gang Li³  Yan-Hong Li³  Guang-Hao Su³  Na-Na Wang³  Zhi-Heng Li³  Li-Chao Sun¹  Xiao-Juan Du²  Pei-Fang Xiao³  Lan Cao³  Fang Fang³  Shao-Yan Hu³  Jun Lu³  Li-Xiao Xu³  Yan-Fang Tao³ 

[1] Department of Cell and Molecular Biology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China;Department of Gastroenterology, the 5th Hospital of Chinese PLA, Yin chuan, China;Department of Hematology and Oncology, Children’s Hospital of Soochow University, Suzhou, China;Translational Research Center, Second Hospital, The Second Clinical School, Nanjing Medical University, Nanjing, China

关键词: Real-time PCR array;    Tumor suppressor;    Methylation;    Pediatric acute myeloid leukemia;    Early B-cell factor 3;   

Others  :  1133647
DOI  :  10.1186/s13046-014-0118-1

 received in 2014-09-15, accepted in 2014-11-27,  发布年份 2015

【 摘 要 】

Background

Pediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear.

Methods

Twelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis.

Results

EBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells.

Conclusion

In this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details.

Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.

【 授权许可】

   
2015 Tao et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Appelbaum FR, Baer MR, Carabasi MH, Coutre SE, Erba HP, Estey E, et al.: NCCN practice guidelines for acute myelogenous leukemia. Oncology (Williston Park) 2000, 14:53-61.
• [2]Sanz GF, Sanz MA, Vallespi T, Canizo MC, Torrabadella M, Garcia S, et al.: Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 1989, 74:395-408.
• [3]Wan H, Zhu J, Chen F, Xiao F, Huang H, Han X, et al.: SLC29A1 single nucleotide polymorphisms as independent prognostic predictors for survival of patients with acute myeloid leukemia: an in vitro study. J Exp Clin Cancer Res 2014, 33:90. BioMed Central Full Text
• [4]Im AP, Sehgal AR, Carroll MP, Smith BD, Tefferi A, Johnson DE, et al.: DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies. Leukemia 2014, 28(9):1774-83.
• [5]Abdel-Wahab O, Levine RL: Mutations in epigenetic modifiers in the pathogenesis and therapy of acute myeloid leukemia. Blood 2013, 121:3563-72.
• [6]Graubert T, Walter MJ: Genetics of myelodysplastic syndromes: new insights. Hematology Am Soc Hematol Educ Program 2011, 2011:543-9.
• [7]Bacher U, Haferlach C, Schnittger S, Kohlmann A, Kern W, Haferlach T: Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies. Ann Hematol 2010, 89:643-52.
• [8]Abdel-Wahab O, Patel J, Levine RL: Clinical implications of novel mutations in epigenetic modifiers in AML. Hematol Oncol Clin North Am 2011, 25:1119-33.
• [9]Larsson CA, Cote G, Quintas-Cardama A: The changing mutational landscape of acute myeloid leukemia and myelodysplastic syndrome. Mol Cancer Res 2013, 11:815-27.
• [10]Schoofs T, Muller-Tidow C: DNA methylation as a pathogenic event and as a therapeutic target in AML. Cancer Treat Rev 2011, 37(Suppl 1):S13-8.
• [11]Conway O’Brien E, Prideaux S, Chevassut T: The epigenetic landscape of acute myeloid leukemia. Adv Hematol 2014, 2014:103175.
• [12]Berenstein R, Blau IW, Kar A, Cay R, Sindram A, Seide C, et al.: Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia. J Exp Clin Cancer Res 2014, 33:44. BioMed Central Full Text
• [13]Melnick AM: Epigenetics in AML. Best Pract Res Clin Haematol 2010, 23:463-8.
• [14]Mann MR, Bartolomei MS: Epigenetic reprogramming in the mammalian embryo: struggle of the clones. Genome Biol 2002, 3(2):REVIEWS1003. BioMed Central Full Text
• [15]Calvo X, Nomdedeu M, Navarro A, Tejero R, Costa D, Munoz C, et al.: High levels of global DNA methylation are an independent adverse prognostic factor in a series of 90 patients with de novo myelodysplastic syndrome. Leuk Res 2014, 38(8):874-81.
• [16]Pleyer L, Burgstaller S, Girschikofsky M, Linkesch W, Stauder R, Pfeilstocker M, et al.: Azacitidine in 302 patients with WHO-defined acute myeloid leukemia: results from the Austrian Azacitidine Registry of the AGMT-Study Group. Ann Hematol 2014, 93(11):1825-38.
• [17]Daskalakis M, Blagitko-Dorfs N, Hackanson B: Decitabine. Recent Results Cancer Res 2010, 184:131-57.
• [18]Hagman J, Ramirez J, Lukin K: B lymphocyte lineage specification, commitment and epigenetic control of transcription by early B cell factor 1. Curr Top Microbiol Immunol 2012, 356:17-38.
• [19]Lukin K, Fields S, Hartley J, Hagman J: Early B cell factor: regulator of B lineage specification and commitment. Semin Immunol 2008, 20:221-7.
• [20]Green YS, Vetter ML: EBF proteins participate in transcriptional regulation of Xenopus muscle development. Dev Biol 2011, 358:240-50.
• [21]Gururajan M, Simmons A, Dasu T, Spear BT, Calulot C, Robertson DA, et al.: Early growth response genes regulate B cell development, proliferation, and immune response. J Immunol 2008, 181:4590-602.
• [22]Fields S, Ternyak K, Gao H, Ostraat R, Akerlund J, Hagman J: The ‘zinc knuckle’ motif of early B cell factor is required for transcriptional activation of B cell-specific genes. Mol Immunol 2008, 45:3786-96.
• [23]Hirokawa S, Sato H, Kato I, Kudo A: EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells. Eur J Immunol 2003, 33:1824-9.
• [24]Liao D: Emerging roles of the EBF family of transcription factors in tumor suppression. Mol Cancer Res 2009, 7:1893-901.
• [25]Zhao LY, Niu Y, Santiago A, Liu J, Albert SH, Robertson KD, et al.: An EBF3-mediated transcriptional program that induces cell cycle arrest and apoptosis. Cancer Res 2006, 66:9445-52.
• [26]Kim J, Min SY, Lee HE, Kim WH: Aberrant DNA methylation and tumor suppressive activity of the EBF3 gene in gastric carcinoma. Int J Cancer 2012, 130:817-26.
• [27]Inaba T: Epidemiology of leukemia and MDS among atomic bomb survivors in Hiroshima and Nagasaki suggests how abnormal epigenetic regulation contributes to leukemogenesis. Rinsho Ketsueki 2009, 50:1548-52.
• [28]Wen-Li Z, Jian W, Yan-Fang T, Xing F, Yan-Hong L, Xue-Ming Z, et al.: Inhibition of the ecto-beta subunit of F1F0-ATPase inhibits proliferation and induces apoptosis in acute myeloid leukemia cell lines. J Exp Clin Cancer Res 2012, 31:92. BioMed Central Full Text
• [29]Olek A, Oswald J, Walter J: A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res 1996, 24:5064-6.
• [30]Cheng Y, Geng H, Cheng SH, Liang P, Bai Y, Li J, et al.: KRAB zinc finger protein ZNF382 is a proapoptotic tumor suppressor that represses multiple oncogenes and is commonly silenced in multiple carcinomas. Cancer Res 2010, 70:6516-26.
• [31]Yan-Fang T, Jian N, Jun L, Na W, Pei-Fang X, Wen-Li Z, et al.: The promoter of miR-663 is hypermethylated in Chinese pediatric acute myeloid leukemia (AML). BMC Med Genet 2013, 14:74. BioMed Central Full Text
• [32]Jian P, Li ZW, Fang TY, Jian W, Zhuan Z, Mei LX, et al.: Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663. J Hematol Oncol 2011, 4:20. BioMed Central Full Text
• [33]Park SH, Kim SK, Choe JY, Moon Y, An S, Park MJ, et al.: Hypermethylation of EBF3 and IRX1 genes in synovial fibroblasts of patients with rheumatoid arthritis. Mol Cells 2013, 35:298-304.
• [34]Bennett KL, Karpenko M, Lin MT, Claus R, Arab K, Dyckhoff G, et al.: Frequently methylated tumor suppressor genes in head and neck squamous cell carcinoma. Cancer Res 2008, 68:4494-9.
• [35]Hong SM, Omura N, Vincent A, Li A, Knight S, Yu J, et al.: Genome-wide CpG island profiling of intraductal papillary mucinous neoplasms of the pancreas. Clin Cancer Res 2012, 18:700-12.
• [36]Yan-Fang T, Dong W, Li P, Wen-Li Z, Jun L, Na W, et al.: Analyzing the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays. Cancer Cell Int 2012, 12:40. BioMed Central Full Text
• [37]Tao YF, Lu J, Du XJ, Sun LC, Zhao X, Peng L, et al.: Survivin selective inhibitor YM155 induce apoptosis in SK-NEP-1 Wilms tumor cells. BMC Cancer 2012, 12:619. BioMed Central Full Text
• [38]Haddick PC, Tom I, Luis E, Quinones G, Wranik BJ, Ramani SR, et al.: Defining the ligand specificity of the deleted in colorectal cancer (DCC) receptor. PLoS One 2014, 9:e84823.
• [39]Meimei L, Peiling L, Baoxin L, Changmin L, Rujin Z, Chunjie H: Lost expression of DCC gene in ovarian cancer and its inhibition in ovarian cancer cells. Med Oncol 2011, 28:282-9.
• [40]Wu M, Xu LG, Li X, Zhai Z, Shu HB: AMID, an apoptosis-inducing factor-homologous mitochondrion-associated protein, induces caspase-independent apoptosis. J Biol Chem 2002, 277:25617-23.
• [41]Marshall KR, Gong M, Wodke L, Lamb JH, Jones DJ, Farmer PB, et al.: The human apoptosis-inducing protein AMID is an oxidoreductase with a modified flavin cofactor and DNA binding activity. J Biol Chem 2005, 280:30735-40.
• [42]Inbal B, Shani G, Cohen O, Kissil JL, Kimchi A: Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis. Mol Cell Biol 2000, 20:1044-54.