Molecular Biology, Pathobiology, and Genetics ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors Marco Ciró, 1 Elena Prosperini, 1 Micaela Quarto, 2 Ursula Grazini, 1 Julian Walfridsson, 3,4 Fraser McBlane, 1 Paolo Nucifero, 2 Giovanni Pacchiana, 1 Maria Capra, 2 Jesper Christensen, 3,4 and Kristian Helin 1,3,4 1 Department of Experimental Oncology, European Institute of Oncology; 2 FIRC Institute of Molecular Oncology, Milan, Italy; and 3 Biotech Research and Innovation Centre and 4 Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark Abstract The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of hu- man cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24,4.3Mbdistalto MYC,inaregionthatisfrequentlyfound amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the ex- pression levels correlate with clinical outcome of breast can- cer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent tran- scription. [Cancer Res 2009;69(21):8491–8] Introduction The retinoblastoma protein (pRB) pathway has a central role in the control of cell proliferation (1). In fact, most human cancers harbor mutations in core members of the pRB pathway, and as a consequence, E2F-regulated genes are aberrantly expressed. Inter- estingly, E2F target genes such as MYC, CCNE1 (cyclin E1), B-MYB, c-MYB, and EZH2 are also bona fide oncogenes, frequently ampli- fied and overexpressed in primary human tumors (2–5). MYC is one of the most studied oncogenes, which contributes to the malignancy of many different aggressive and undifferentiated human cancers (4). The pathologic effect of MYC has been ascribed to its ability to control many cellular processes such as cell growth, differentiation, apoptosis, and, more recently, DNA damage re- sponse, genomic instability, angiogenesis, and tumor invasiveness (6). MYC acts as a potent, sequence-specific transcription factor (6) interacting both with the SWI/SNF chromatin remodeling com- plexes (7) and with the histone acetyltransferases CBP/p300 (8), GCN5 (9, 10), and TIP60 (11). Here we report on a hitherto uncharacterized E2F target gene, ATAD2. ATAD2 is highly expressed and genetically amplified in sev- eral types of human cancer, and we show that high levels of ATAD2 correlate with unfavorable prognosis in breast cancer patients. Furthermore, we show that ATAD2 works as an important cofactor for MYC-dependent transcription, suggesting that ATAD2 contri- butes to tumor development, in part, through enhancing the tran- scriptional activity of MYC. Materials and Methods Tissue culture. Cell lines were grown in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 units/mL streptomycin. Transient transfections were done with stan- dard CaPO 4 method. Crystal violet staining and quantification were done as described (12). HeLa cells were synchronized by double-thymidine block (13) and released from the block in the presence of 100 ng/mL nocodazole. RNA interference. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) against ATAD2 were generated targeting two different se- quences: GGATCTCTCTTCAATTAAT and GTGCGTCGAAGTTGTAGGA, respectively. SiRNA duplexes from annealed pairs of 21 ribonucleotides (Dharmacon Research) were transfected into TIG3 fibroblasts using Oligo- fectamine (Invitrogen). ShRNAs were expressed from lentiviruses generated using pLL3.7 vector (14). As a control, we used siRNA or shRNA duplex targeting firefly luciferase. Antibodies. We raised and affinity purified a polyclonal antibody against ATAD2 by immunizing rabbits with a MBP NH 2 -terminal portion of the protein (amino acids 1–447). In addition, the following antibodies were used: anti-vinculin (h-VIN1, Sigma), anti-HA (HA.11, BABCO), anti- FLAG (M2, Sigma), anti-CCNA2 (C-19, Santa Cruz Biotechnology), anti-MYC (N-262, C-33 Santa Cruz Biotechnology), mouse anti-bromodeoxyuridine (BrdUrd; Becton Dickinson), anti-E2F1 (KH20; ref. 15), anti-H3 (ab1791, Abcam), anti–acetylated H3 (Upstate), anti–α-tubulin (DM-1A, Sigma), anti –lamin A/C (sc-7292, Santa Cruz Biotechnology), anti –cyclin E1 (M20, Santa Cruz Biotechnology), anti-TIP60 (11), and cyclin B1 (H-20, Santa Cruz Biotechnology). Antibodies used in chromatin immunopre- cipitation assays are described below. Chromatin immunoprecipitation. Chromatin immunoprecipitations were done and analyzed essentially as described (16). The antibodies used were immunopurified polyclonal anti-ATAD2, anti-MYC (N-262, Santa Cruz Biotechnology),anti-E2F1,anti-E2F2,anti-E2F3(sc-193,sc-633,sc-878,Santa Cruz Biotechnology), and anti-HA (HA.11, BABCO) as a control. Primer sequences for amplification of ATAD2 promoter are available on request. Primer sequences for amplification of MYC target promoters and acetyl- cholinereceptorpromoterwereobtainedfromFernandezandcolleagues.(17). Sequential nuclear extraction. Sequential nuclear extraction was done as described in ref. 18. In the first fractionation step, soluble proteins were removed by extraction with Triton X-100. Chromatin proteins were then released by DNase I digestion followed by 0.25 mol/L ammonium sulfate treatment, which resulted in the release of most of the histones. After a 2 mol/L NaCl wash, the last fraction was enriched in nuclear matrix– associated proteins. Recombinant protein production. Recombinantglutathione S-transferase (GST)-ATAD2 was obtained from sf9 cells as previously described (19). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Kristian Helin, Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaloes Vej 5, Copenhagen 2200, Denmark. Phone: 45-3532-5666; Fax: 45-3532-5669; E-mail: kristian.helin@bric.ku.dk. ©2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-2131 8491 Cancer Res 2009; 69: (21). November 1, 2009 www.aacrjournals.org Research. on July 27, 2017. © 2009 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst October 20, 2009; DOI: 10.1158/0008-5472.CAN-09-2131