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.
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.
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