A REST derived gene signature stratifies glioblastomas into chemotherapy resistant and responsive disease
© Wagoner and Roopra; licensee BioMed Central Ltd. 2012
Received: 18 July 2012
Accepted: 28 November 2012
Published: 7 December 2012
Glioblastomas are the most common central nervous system neoplasia in adults, with 9,000 cases in the US annually. Glioblastoma multiformae, the most aggressive glioma subtype, has an 18% one-year survival rate, and 3% two year survival rate. Recent work has highlighted the role of the transcription factor RE1 Silencing Transcription Factor, REST in glioblastoma but how REST function correlates with disease outcome has not been described.
Using a bioinformatic approach and mining of publicly available microarray datasets, we describe an aggressive subtype of gliomas defined by a gene signature derived from REST. Using this REST gene signature we predict that REST function is enhanced in advanced glioblastoma. We compare disease outcomes between tumors based on REST status and treatment regimen, and describe downstream targets of REST that may contribute to the decreased benefits observed with high dose chemotherapy in REM tumors.
We present human data showing that patients with “REST Enhanced Malignancies” (REM) tumors present with a shorter disease free survival compared to non-REM gliomas. Importantly, REM tumors are refractory to multiple rounds of chemotherapy and patients fail to respond to this line of treatment.
This report is the first to describe a REST gene signature that predicts response to multiple rounds of chemotherapy, the mainline therapy for this disease. The REST gene signature may have important clinical implications for the treatment of glioblastoma.
KeywordsREST RE1 NRSE NRSF Glioblastoma Patient data Chemotherapy Bioinformatics
RE1-silencing transcription factor (REST) is a transcriptional repressor that regulates the expression of approximately 2,000 neuronal genes in neural and non-neural tissues, including embryonic and neural stem cells (NSC) [1–4]. In development, loss of REST function is an integral step in NSC differentiation, and inappropriate maintenance of REST function has been found to prevent differentiation of NSC into neurons [4–6]. In neoplasia, heightened REST function in medulloblastoma tumor cells contributes to their tumorigenicity in mouse models of the disease, in part by preventing their differentiation [7, 8]. Accordingly, many human medulloblastoma tumors show significantly higher REST protein levels than adjacent normal brain tissue. Increased REST levels also correlated with lower overall and event-free survival .
Glioblastomas are the most common central nervous system neoplasia in adults, with 9,000 cases in the US annually. Regardless of whether gliomas arise from astrocytes (astrocytomas) or oligodendrocytes (oligodendromas), they carry with them a uniformly poor prognosis. Glioblastoma multiformae, the most aggressive glioma subtype, has an 18% one-year survival rate, and 3% two-year survival rate . Recently, a role for REST was suggested in glioma with REST up-regulation able to drive cell proliferation and suppress differentiation [11, 12]. Elegant work from Conti et al and Kamal et al showed increased REST protein in glioblastoma samples versus control brain tissue and knockdown of REST in glioblastoma cell lines reduced proliferation rate and promoted differentiation. However, how REST levels corresponded to patient outcome was not described. In this work, we will describe an aggressive subtype of gliomas with enhanced REST function, termed REST Enhanced Malignancies (REM). We compare disease outcomes between tumors based on REST status and treatment regimen, and describe downstream targets of REST that may contribute to the decreased benefits observed with high dose chemotherapy in REM tumors.
Results and discussion
Evaluation of neuronal non-REST target genes found that there was no concerted up-regulation of all neuronal non-REST markers in either REM or near-normal tumors. Similarly, Vascular endothelial markers VEGFR and VE-Cadherin were not enriched in REM tumors (data not shown) Thus we conclude that different levels of neuronal or vascular involvement between tumors are not responsible for the observed variation in REST function (Figure 4B and C).
Glioma tumor suppressors [Roopra]
RasGAP tumorsuppressor. Loss of NF1 by mutation or degradation occurs in many gliomas and is associated with chemotherapy resistance
Suppresses growth of glioma xenografts, reintroduction of BEX1 in glioma cell lines induces chemotherapy sensitivity
Glioma tumor suppressor known to regulate cell growth, apaptosis and sensitivity to chemotherapy
Glioma tumor suppressor that regulates proliferation and differentiation
BEX1 is a glioma tumor suppressor gene, the overexpression of which effectively suppresses human glioma xenograft tumor growth in nude mice . BEX1 mRNA expression is lost many gliomas, in part through promoter methylation . Published ChIP-Seq analysis for REST bound genes found that REST directly binds the BEX1 gene in Jurkat T-cells, suggesting that it too is an endogenous REST target . BEX1 mRNA shows a strong correlation with REST signature gene expression (p<10-16) and is two-fold lower in REM tumors than near-normal tumors, suggesting that the reduced BEX1 expression observed in these tumors may be due to increased REST function.
p27KIP1 is a cyclin dependent kinase inhibitor that regulates the G1/S transition by inhibiting a number of CDK complexes, including CDK2 and CDK4 . Decreased expression of p27KIP1 in astrocytomas is associated with increased proliferation, and decreased patient survival [31, 32]. p27KIP1 mRNA levels in tumors correlate with REST signature gene expression (p<10-9) and its gene contains a consensus REST binding site, suggesting that the reduced p27KIP1 expression observed in these tumors may be due to increased REST function.
Interestingly, loss of NF1, p27KIP1 and BEX1 are all associated with glioma chemotherapy resistance [30, 31, 33, 34], suggesting that these genes may play a role in the reduced benefit of high dose chemotherapy in patients with REM tumors.
Here, we have provided evidence that REST function is increased in glioma tumors and that this heightened activity correlates with differential tumor aggressiveness and response to treatment. Our findings suggest a mechanism by which REST function may be enhanced in gliomas via loss of β-TrCP expression.
Importantly, we show that a decrease in a specific suite of REST target genes correlates with failure to respond to multiple round of chemotherapy, a finding of significant clinical impact.
Transcriptional analyses on the microarray data were performed using BRB-ArrayTools v3.7 (developed by Dr. Richard Simon and BRB-ArrayTools Development Team) and MultiExperiment Viewer 4.5.1. Tumor gene expression data were obtained from the NCBI Gene Expression Omnibus, and are identified by their GEO dataset record number, with the exception of the cancer genome atlas (TCGA) dataset, which was not available on GEO at the time of manuscript submission. TCGA datasets are described . Hierarchical clustering was performed using a one-minus correlation metric with average linkage over centered genes. Cluster diagrams were produced using BRB Arraytools, Cluster 3.0 and TreeView software.
The consensus clustering method was used to determine how many REST-activity delimited glioma subgroups may be reproducibly established in an unbiased fashion. First, genes that showed a high correlation of expression with the REST 24-gene signature at p<10-8 were defined using Pavlidis Template Matching using the MultiExperiment Viewer platform using the 200 tumor TCGA dataset. From this, 403 genes were identified and subjected to Consensus Clustering, which was performed using BRB array tools. One thousand iterations were used to classify tumors into 2, 3, 4 and 5 REST subtypes. In subsequent analyses, this analysis was used to classify tumors into just 3 REST-activity based subtypes.
Gene set enrichment analysis
Gene Set Enrichment Analysis (GSEA) was performed using the GSEA program provided by the Broad Institute. The list of genes identified as likely REST targets were identified in Johnson et al. using ChIP-Seq with an anti-REST antibody. Genes were determined to be likely REST targets based on their ChIP-Seq enrichment in two independent experiments in a region carrying an RE1 site with a p-value of < 10-4.
Patient survival curves were generated using PRISM and MSTAT software (http://www.mcardle.wisc.edu/mstat/).
Molecular classification comparison
Molecular classification of glioma tumors into classical, mesenchymal, proneural and neural subtype information for the TCGA tumor samples was published in Verhaak et al . To determine if tumor stratification by REST activity level overlapped significantly with established molecular classifications, these same tumors were re-classified using the consensus clustering method described above and co-incidence of classification is indicated both with respect to published molecular subtype (top) and REST activity level (bottom).
Copy number analysis
Copy number analysis was performed using integrative genomics viewer from the Broad Institute (IGV - http://www.broadinstitute.org/igv/home). IGV was used to assess copy number variations in 141 glioma tumors in dataser GSE9635 previously published and characterized .
We would like to thank members of the Roopra lab for advice with the manuscript. This work was supported by an NIH/NINDS grant to A.R. (NS065067) and the Dual Sports Riders of Wisconsin. M.P.W. was supported by Molecular and Cellular Pharmacology program.
- Chong JA, Tapia-Ramirez J, Kim S, Toledo-Aral JJ, Zheng Y, Boutros MC, Altshuller YM, Frohman MA, Kraner SD, Mandel G: REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell. 1995, 80 (6): 949-957. 10.1016/0092-8674(95)90298-8.View ArticlePubMedGoogle Scholar
- Schoenherr CJ, Anderson DJ: The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science. 1995, 267 (5202): 1360-1363. 10.1126/science.7871435.View ArticlePubMedGoogle Scholar
- Johnson DS, Mortazavi A, Myers RM, Wold B: Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007, 316 (5830): 1497-1502. 10.1126/science.1141319.View ArticlePubMedGoogle Scholar
- Johnson R, Teh CH, Kunarso G, Wong KY, Srinivasan G, Cooper ML, Volta M, Chan SS, Lipovich L, Pollard SM, et al: REST regulates distinct transcriptional networks in embryonic and neural stem cells. PLoS Biol. 2008, 6 (10): e256-10.1371/journal.pbio.0060256.PubMed CentralView ArticlePubMedGoogle Scholar
- Guardavaccaro D, Frescas D, Dorrello NV, Peschiaroli A, Multani AS, Cardozo T, Lasorella A, Iavarone A, Chang S, Hernando E, et al: Control of chromosome stability by the beta-TrCP-REST-Mad2 axis. Nature. 2008, 452 (7185): 365-369. 10.1038/nature06641.PubMed CentralView ArticlePubMedGoogle Scholar
- Lessard J, Wu JI, Ranish JA, Wan M, Winslow MM, Staahl BT, Wu H, Aebersold R, Graef IA, Crabtree GR: An essential switch in subunit composition of a chromatin remodeling complex during neural development. Neuron. 2007, 55 (2): 201-215. 10.1016/j.neuron.2007.06.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Fuller GN, Su X, Price RE, Cohen ZR, Lang FF, Sawaya R, Majumder S: Many human medulloblastoma tumors overexpress repressor element-1 silencing transcription (REST)/neuron-restrictive silencer factor, which can be functionally countered by REST-VP16. Mol Canc Ther. 2005, 4 (3): 343-349.Google Scholar
- Lawinger P, Venugopal R, Guo ZS, Immaneni A, Sengupta D, Lu W, Rastelli L, Marin Dias Carneiro A, Levin V, Fuller GN, et al: The neuronal repressor REST/NRSF is an essential regulator in medulloblastoma cells. Nat Med. 2000, 6 (7): 826-831. 10.1038/77565.View ArticlePubMedGoogle Scholar
- Taylor P, Fangusaro J, Rajaram V, Goldman S, Helenowski IB, MacDonald T, Hasselblatt M, Riedemann L, Laureano A, Cooper L, et al: REST is a novel prognostic factor and therapeutic target for medulloblastoma. Mol Canc Ther. 2012, 11 (8): 1713-1723. 10.1158/1535-7163.MCT-11-0990.View ArticleGoogle Scholar
- Ohgaki H, Dessen P, Jourde B, Horstmann S, Nishikawa T, Di Patre PL, Burkhard C, Schuler D, Probst-Hensch NM, Maiorka PC, et al: Genetic pathways to glioblastoma: a population-based study. Canc Res. 2004, 64 (19): 6892-6899. 10.1158/0008-5472.CAN-04-1337.View ArticleGoogle Scholar
- Conti L, Crisafulli L, Caldera V, Tortoreto M, Brilli E, Conforti P, Zunino F, Magrassi L, Schiffer D, Cattaneo E: REST Controls Self-Renewal and Tumorigenic Competence of Human Glioblastoma Cells. PLoS One. 2012, 7 (6): e38486-10.1371/journal.pone.0038486.PubMed CentralView ArticlePubMedGoogle Scholar
- Kamal MM, Sathyan P, Singh SK, Zinn PO, Marisetty AL, Liang S, Gumin J, El-Mesallamy HO, Suki D, Colman H, et al: REST regulates oncogenic properties of glioblastoma stem cells. Stem Cells. 2012, 30 (3): 405-414. 10.1002/stem.1020.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagoner MP, Gunsalus KTW, Schoenike B, Richardson AL, Friedl A, Roopra A: The Transcription Factor REST Is Lost in Aggressive Breast Cancer. PLoS Genet. 2010, 6 (6): e1000979-10.1371/journal.pgen.1000979.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun L, Hui AM, Su Q, Vortmeyer A, Kotliarov Y, Pastorino S, Passaniti A, Menon J, Walling J, Bailey R, et al: Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Canc Cell. 2006, 9 (4): 287-300. 10.1016/j.ccr.2006.03.003.View ArticleGoogle Scholar
- Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, et al: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102 (43): 15545-15550. 10.1073/pnas.0506580102.PubMed CentralView ArticlePubMedGoogle Scholar
- Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, et al: A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. 2008, 28 (1): 264-278. 10.1523/JNEUROSCI.4178-07.2008.View ArticlePubMedGoogle Scholar
- Abrajano JJ, Qureshi IA, Gokhan S, Zheng D, Bergman A, Mehler MF: Differential deployment of REST and CoREST promotes glial subtype specification and oligodendrocyte lineage maturation. PLoS One. 2009, 4 (11): e7665-10.1371/journal.pone.0007665.PubMed CentralView ArticlePubMedGoogle Scholar
- Beroukhim R, Getz G, Nghiemphu L, Barretina J, Hsueh T, Linhart D, Vivanco I, Lee JC, Huang JH, Alexander S, et al: Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proc Natl Acad Sci USA. 2007, 104 (50): 20007-20012. 10.1073/pnas.0710052104.PubMed CentralView ArticlePubMedGoogle Scholar
- Ballas N, Grunseich C, Lu DD, Speh JC, Mandel G: REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. Cell. 2005, 121 (4): 645-657. 10.1016/j.cell.2005.03.013.View ArticlePubMedGoogle Scholar
- Westbrook TF, Hu G, Ang XL, Mulligan P, Pavlova NN, Liang A, Leng Y, Maehr R, Shi Y, Harper JW, et al: SCFbeta-TRCP controls oncogenic transformation and neural differentiation through REST degradation. Nature. 2008, 452 (7185): 370-374. 10.1038/nature06780.PubMed CentralView ArticlePubMedGoogle Scholar
- Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, Miller CR, Ding L, Golub T, Mesirov JP, et al: Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Canc Cell. 2010, 17 (1): 98-110. 10.1016/j.ccr.2009.12.020.View ArticleGoogle Scholar
- Monti S, Tamayo P, Mesirov J, Golub TR: Consensus Clustering: a resampling-based method for class discovery and visualization of gene expression microarray data. Mach Learn. 2003, 52: 91-118. 10.1023/A:1023949509487.View ArticleGoogle Scholar
- Buyse M, Loi S, van’t Veer L, Viale G, Delorenzi M, Glas AM, d’Assignies MS, Bergh J, Lidereau R, Ellis P, et al: Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J Natl Canc Inst. 2006, 98 (17): 1183-1192. 10.1093/jnci/djj329.View ArticleGoogle Scholar
- Kelly CM, Krishnamurthy S, Bianchini G, Litton JK, Gonzalez-Angulo AM, Hortobagyi GN, Pusztai L: Utility of oncotype DX risk estimates in clinically intermediate risk hormone receptor-positive, HER2-normal, grade II, lymph node-negative breast cancers. Cancer. 2010, 116 (22): 5161-5167. 10.1002/cncr.25269.View ArticlePubMedGoogle Scholar
- Alexiou GA, Voulgaris S: The role of the PTEN gene in malignant gliomas. Neurol Neurochir Pol. 2010, 44 (1): 80-86.PubMedGoogle Scholar
- Freije WA, Castro-Vargas FE, Fang Z, Horvath S, Cloughesy T, Liau LM, Mischel PS, Nelson SF: Gene expression profiling of gliomas strongly predicts survival. Canc Res. 2004, 64 (18): 6503-6510. 10.1158/0008-5472.CAN-04-0452.View ArticleGoogle Scholar
- Conaco C, Han JJ, Mandel G: Reciprocal actions of REST and a microRNA promote neuronal identity. Proc Natl Acad Sci USA. 2006, 103 (7): 2422-7. 10.1073/pnas.0511041103.PubMed CentralView ArticlePubMedGoogle Scholar
- Bernardini M, Lee CH, Beheshti B, Prasad M, Albert M, Marrano P, Begley H, Shaw P, Covens A, Murphy J, et al: High-resolution mapping of genomic imbalance and identification of gene expression profiles associated with differential chemotherapy response in serous epithelial ovarian cancer. Neoplasia. 2005, 7 (6): 603-613. 10.1593/neo.04760.PubMed CentralView ArticlePubMedGoogle Scholar
- Purow B, Schiff D: Advances in the genetics of glioblastoma: are we reaching critical mass?. Nat Rev Neurol. 2009, 5 (8): 419-426. 10.1038/nrneurol.2009.96.PubMed CentralView ArticlePubMedGoogle Scholar
- Foltz G, Ryu GY, Yoon JG, Nelson T, Fahey J, Frakes A, Lee H, Field L, Zander K, Sibenaller Z, et al: Genome-wide analysis of epigenetic silencing identifies BEX1 and BEX2 as candidate tumor suppressor genes in malignant glioma. Canc Res. 2006, 66 (13): 6665-6674. 10.1158/0008-5472.CAN-05-4453.View ArticleGoogle Scholar
- Cheney IW, Neuteboom ST, Vaillancourt MT, Ramachandra M, Bookstein R: Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin E/CDK2 complexes. Canc Res. 1999, 59 (10): 2318-2323.Google Scholar
- Fuse T, Tanikawa M, Nakanishi M, Ikeda K, Tada T, Inagaki H, Asai K, Kato T, Yamada K: p27Kip1 expression by contact inhibition as a prognostic index of human glioma. J Neurochem. 2000, 74 (4): 1393-1399.View ArticlePubMedGoogle Scholar
- Naumann U, Weit S, Rieger L, Meyermann R, Weller M: p27 modulates cell cycle progression and chemosensitivity in human malignant glioma. Biochem Biophys Res Commun. 1999, 261 (3): 890-896. 10.1006/bbrc.1999.1126.View ArticlePubMedGoogle Scholar
- Shapira S, Barkan B, Friedman E, Kloog Y, Stein R: The tumor suppressor neurofibromin confers sensitivity to apoptosis by Ras-dependent and Ras-independent pathways. Cell Death Differ. 2007, 14 (5): 895-906.PubMedGoogle Scholar
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