MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

BMC Cancer

Leleesha Samaraweera¹  Kathryn B Grandinetti³  Ruojun Huang²  Barbara A Spengler⁴  Robert A Ross⁴ 

[1] Albert Einstein College of Medicine, 1300, Morris Park Ave, Bronx, NY 10461, USA

[2] Edison, NJ, USA

[3] Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA

[4] Fordham University, 441 E. Fordham Road, Bronx, NY 10458, USA

关键词: HuD;    N-myc;    miR-124;    miR-375;    MicroRNAs;    Tumorigenicity;    Differentiation;    Neuroblastoma;   

Others  :  858849
DOI  :  10.1186/1471-2407-14-309

 received in 2013-11-19, accepted in 2014-04-11,  发布年份 2014

【 摘 要 】

Background

Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties.

Methods

A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation.

Results

Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD.

Conclusions

Thus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer.

【 授权许可】

   
2014 Samaraweera et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Park JR, Bagatell R, London WB, Maris JM, Cohn SL, Mattay KM, Hogarty M, Committee COGN: Children’s Oncology Group’s 2013 blueprint for research: neuroblastoma. Pediatric Blood Cancer 2013, 60(6):985-993.
• [2]Ross RA, Spengler BA: Human neuroblastoma stem cells. Semin Cancer Biol 2007, 17(3):241-247.
• [3]Spengler BA, Lazarova DL, Ross RA, Biedler JL: Cell lineage and differentiation state are primary determinants of MYCN gene expression and malignant potential in human neuroblastoma cells. Oncol Res 1997, 9(9):467-476.
• [4]Walton JD, Kattan DR, Thomas SK, Spengler BA, Guo HF, Biedler JL, Cheung NK, Ross RA: Characteristics of stem cells from human neuroblastoma cell lines and in tumors. Neoplasia 2004, 6(6):838-845.
• [5]Shimada H, Chatten J, Newton WA Jr, Sachs N, Hamoudi AB, Chiba T, Marsden HB, Misugi K: Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J Natl Cancer Inst 1984, 73(2):405-416.
• [6]Eggert A, Schulte JH: A small kiss of death for cancer. Nat Med 2010, 16(10):1079-1081.
• [7]Stallings RL: MicroRNA involvement in the pathogenesis of neuroblastoma: potential for microRNA mediated therapeutics. Curr Pharm Des 2009, 15(4):456-462.
• [8]Stallings RL, Foley NH, Bryan K, Buckley PG, Bray I: Therapeutic targeting of miRNAs in neuroblastoma. Expert Opin Ther Targets 2010, 14(9):951-962.
• [9]Stallings RL, Foley NH, Bray IM, Das S, Buckley PG: MicroRNA and DNA methylation alterations mediating retinoic acid induced neuroblastoma cell differentiation. Semin Cancer Biol 2011, 21(4):283-290.
• [10]Ikegaki N, Bukovsky J, Kennett RH: Identification and characterization of the NMYC gene product in human neuroblastoma cells by monoclonal antibodies with defined specificities. Proc Natl Acad Sci U S A 1986, 83(16):5929-5933.
• [11]Thomas SK, Messam CA, Spengler BA, Biedler JL, Ross RA: Nestin is a potential mediator of malignancy in human neuroblastoma cells. J Biol Chem 2004, 279(27):27994-27999.
• [12]Schulte JH, Horn S, Otto T, Samans B, Heukamp LC, Eilers UC, Krause M, Astrahantseff K, Klein-Hitpass L, Buettner R, Schramm A, Christiansen H, Eilers M, Eggert A, Berwanger B: MYCN regulates oncogenic MicroRNAs in neuroblastoma. Int J Cancer 2008, 122(3):699-704.
• [13]Lin RJ, Lin YC, Chen J, Kuo HH, Chen YY, Diccianni MB, London WB, Chang CH, Yu AL: microRNA signature and expression of Dicer and Drosha can predict prognosis and delineate risk groups in neuroblastoma. Cancer Res 2010, 70(20):7841-7850.
• [14]Howard M, Foster DN, Cserjesi P: Expression of HAND gene products may be sufficient for the differentiation of avian neural crest-derived cells into catecholaminergic neurons in culture. Dev Biol 1999, 215(1):62-77.
• [15]Louvi A, Artavanis-Tsakonas S: Notch signalling in vertebrate neural development. Nature Rev Neurosci 2006, 7(2):93-102.
• [16]Liu XS, Chopp M, Zhang RL, Tao T, Wang XL, Kassis H, Hozeska-Solgot A, Zhang L, Chen C, Zhang ZG: MicroRNA profiling in subventricular zone after stroke: MiR-124a regulates proliferation of neural progenitor cells through Notch signaling pathway. PloS one 2011, 6(8):e23461.
• [17]Guo J, Dong Q, Fang Z, Chen X, Lu H, Wang K, Yin Y, Cai X, Zhao N, Chen J, Zen K, Zhang J, Zhang CY: Identification of miRNAs that are associated with tumor metastasis in neuroblastoma. Cancer Biol Ther 2010, 9(6):446-452.
• [18]Lynch J, Fay J, Meehan M, Bryan K, Watters KM, Murphy DM, Stallings RL: MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-beta signalling pathway. Carcinogenesis 2012, 33(5):976-985.
• [19]Bray I, Bryan K, Prenter S, Buckley PG, Foley NH, Murphy DM, Alcock L, Mestdagh P, Vandesompele J, Speleman F, London WB, McGrady PW, Higgins DG, O’Meara A, O'Sullivan M, Stallings RL: Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival. PLoS One 2009, 4(11):e7850.
• [20]Chen Y, Stallings RL: Differential patterns of microRNA expression in neuroblastoma are correlated with prognosis, differentiation, and apoptosis. Cancer Res 2007, 67(3):976-983.
• [21]Conaco C, Otto S, Han JJ, Mandel G: Reciprocal actions of REST and a microRNA promote neuronal identity. Proc Natl Acad Sci U S A 2006, 103(7):2422-2427.
• [22]Krichevsky AM, Sonntag KC, Isacson O, Kosik KS: Specific microRNAs modulate embryonic stem cell-derived neurogenesis. Stem Cells 2006, 24(4):857-864.
• [23]Mouillet-Richard S, Mutel V, Loric S, Tournois C, Launay JM, Kellermann O: Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation. J Biol Chem 2000, 275(13):9186-9192.
• [24]Thiele CJ, Reynolds CP, Israel MA: Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma. Nature 1985, 313(6001):404-406.
• [25]Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, Swift P, Shimada H, Black CT, Brodeur GM, Gerbing RB, Reynolds CP: Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med 1999, 341(16):1165-1173.
• [26]Le MT, Xie H, Zhou B, Chia PH, Rizk P, Um M, Udolph G, Yang H, Lim B, Lodish HF: MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets. Mol Cell Biol 2009, 29(19):5290-5305.
• [27]Avnit-Sagi T, Kantorovich L, Kredo-Russo S, Hornstein E, Walker MD: The promoter of the pri-miR-375 gene directs expression selectively to the endocrine pancreas. PLoS One 2009, 4(4):e5033.
• [28]Grandinetti KB, Spengler BA, Biedler JL, Ross RA: Loss of one HuD allele on chromosome #1p selects for amplification of the N-myc proto-oncogene in human neuroblastoma cells. Oncogene 2006, 25(5):706-712.
• [29]Kim JW, Zeller KI, Wang Y, Jegga AG, Aronow BJ, O'Donnell KA, Dang CV: Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays. Mol Cell Biol 2004, 24(13):5923-5936.
• [30]Abdelmohsen K, Hutchison ER, Lee EK, Kuwano Y, Kim MM, Masuda K, Srikantan S, Subaran SS, Marasa BS, Mattson MP, Gorospe M: miR-375 inhibits differentiation of neurites by lowering HuD levels. Mol Cell Biol 2010, 30(17):4197-4210.
• [31]Chu VR, Lee I: MicroRNA Target Signatures in Advanced Stage Neuroblastoma. In Neuroblastoma - Present and Future. Edited by Shimada H. Intech; 2012. doi:10.5772/29142