Cancer Genes and Genomics Identification of Genomic Targets of Transcription Factor Aebp1 and its role in Survival of Glioma Cells Jayashree Ladha, Swati Sinha, Vasudeva Bhat, Sainitin Donakonda, and Satyanarayana M.R. Rao Abstract A recent transcriptome analysis of graded patient glioma samples led to identification of AEBP1 as one of the genes upregulated in majority of the primary GBM as against secondary GBM. Aebp1 is a transcriptional repressor that is involved in adipogenesis. It binds to AE-1 element present in the proximal promoter of aP2 gene that codes for fatty acid binding protein (FABP4). A comprehensive study was undertaken to elucidate the role of AEBP1 overexpression in glioblastoma. We employed complementary gene silencing approach to identify the genes that are perturbed in a glioma cell line (U87MG). A total of 734 genes were differentially regulated under these conditions (1.5-fold, P  0.05) belonging to different GO categories such as transcription regulation, cell growth, proliferation, differentiation, and apoptosis of which perturbation of 114 genes of these pathways were validated by quantitative real time PCR (qRT-PCR). This approach was subsequently combined with ChIP-chip technique using an Agilent human promoter tiling array to identify genomic binding loci of Aebp1 protein. A subset of these genes identified for Aebp1 occupancy was also validated by ChIP-PCR. Bioinformatics analysis of the promoters identified by ChIP-chip technique revealed a consensus motif GAAAT present in 66% of the identified genes. This consensus motif was experimentally validated by functional promoter assay using luciferase as the reporter gene. Both cellular proliferation and survival were affected in AEBP1-silenced U87MG and U138MG cell lines and a significant percentage of these cells were directed towards apoptosis. Mol Cancer Res; 1–13. Ó2012 AACR. Introduction Glioblastoma multiforme (GBM) is the most common and malignant form of primary tumor of CNS in adults, which is characterized by a median survival of less than a year. The prognostic behavior of GBMs is rather poor and hence there have been efforts to identify molecular signatures and also to discover new biomarkers for characterizing different types and stages of GBMs (1–4). GBM is broadly classified into primary and secondary GBM (WHO), each one arising through distinct genetic pathways. Primary GBM arises de novo and is frequently associated with amplification and/or overexpression of EGFR and PTEN deletion combined with INK4A/ARF and CDKN2A losses and MDM2 amplification (5). However, secondary GBM often exhibits P53 muta- tions, PDGF/PDGFR overexpression, RB loss, and CDK4 amplifications (6). Recent studies have shown, however, that there is an overlapping spectrum of mutations in these 2 types of GBM (7, 8). In one of our earlier studies we had found AEBP1 expression to be upregulated in primary GBMs as opposed to progressive secondary GBMs (9). Aebp1 was originally identified as a transcriptional repressor that binds to adipocyte enhancer 1 (AE-1 element) located in the proximal promoter region of the adipose P2 gene, which codes for adipocyte specific fatty acid binding protein 4 (FABP4; ref. 10). Aebp1 is also overexpressed in transgenic mouse probasin-Neu (ERBB2) induced advanced prostate cancer (11). However, the exact role of AEBP1 in tumorigenesis is not clear and hence we set out to identify the genomic targets of this transcription factor to understand its biology in the cellular context. Toward this direction we have undertaken a detailed study to analyze the Aebp1 genomic targets by transcriptome profiling of AEBP1 downregulated U87MG cells and its role in cell proliferation, growth, and survival. Materials and Methods Cell culture and AEBP1 silencing U87MG and U138MG cells (ATCC) were grown in Eagle's Minimal Essential Medium supplemented with 10% FBS (Sigma-Aldrich). Cells were transfected with 100 nmol/L siRNA pool targeted against AEBP1 (Dharma- con Inc.). Quantitative real-time PCR (qRT-PCR) was done using Eva Green (Biorad) on a Biorad iQ5 cycler. Down- regulation of AEBP1 was assessed by qRT-PCR and Western Authors' Affiliation: Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). J. Ladha and S. Sinha have made equal contributions to this article. V. Bhat and S. Donakonda have made equal contributions to this article. Corresponding Author: Satyanarayana M.R. Rao, Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India. Phone: +91 9886233032; Fax: +91-80-22082766/23602468; E-mail: mrsrao@jncasr.ac.in doi: 10.1158/1541-7786.MCR-11-0488 Ó2012 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1 Research. on January 21, 2022. © 2012 American Association for Cancer mcr.aacrjournals.org Downloaded from Published OnlineFirst June 20, 2012; DOI: 10.1158/1541-7786.MCR-11-0488