Molecular and Cellular Pathobiology Negative Regulation of p53 by the Long Isoform of ErbB3 Binding Protein Ebp1 in Brain Tumors Chung Kwon Kim 1 , Truong L.X. Nguyen 1 , Kyeung Min Joo 4 , Do-Hyun Nam 5 , Jihye Park 1 , Kyung-Hoon Lee 2,3 , Sung-Woo Cho 6 , and Jee-Yin Ahn 1,3 Abstract The ErbB3 binding protein Ebp1 has been implicated in a number of human cancers. Ebp1 includes 2 isoforms, p48 and p42, that exhibit different cellular activities. Here we show that the larger p48 isoform is transforming and that it promotes cell growth, clonogenicity, and invasion in human glioblastoma (GBM). P48 overexpression in GBM cells facilitated tumorigenesis and enhanced tumor growth in mouse xenograft models. Human GBM tissues displayed elevated levels of p48 compared with surrounding normal tissues or low-grade tumors. Notably, p48 levels were inversely correlated with poor prognosis in GBM patients. We determined that p48 binds to the p53 E3 ligase HDM2, enhancing HDM2-p53 association and thereby promoting p53 polyubiquitination and degradation to reduce steady-state p53 levels and activity. Together, our findings suggest that p48 functions as an oncogene by promoting glioma tumorigenicity via interactions with HDM2 that contribute to p53 down- regulation. Cancer Res; 70(23); 9730–41. Ó2010 AACR. Introduction Ebp1 is the human homologue of the mouse protein p38- 2AG4 (1). The p38-2AG4, PA2G4, possesses 3 in-frame ATG codons and encodes 2 alternative spliced isoforms, p48 and p42, which differentially mediate PC12 cell survival and differ- entiation (2, 3). Recently, we showed that p48 Ebp1 prevents apoptotic cell death by interacting with nuclear Akt/PKB (protein kinase B) (2), fitting with the observation that Ebp1 binds Bcl2 mRNA and contributes to Bcl2 overexpres- sion (4). During the early stages of organ development in plant, stEBP1 promotes cell proliferation, affects the cell size thresh- old, and shortens the period of meristematic activity. In mitotic cells, Ebp1 enhances cell expansion (5). Furthermore, tamoxifen treatment of MCF-7 breast cancer cells dramati- cally decreases p48 Ebp1 transcription and, thus, protein levels. Breast cancer patients that express high level of PA2G4 have poor clinical outcomes, suggesting Ebp1 may promote aggressive tumor behavior (6). Treatment with heregulin induces the nuclear translocation of p42 Ebp1 from the cytoplasm in AU565 breast cancer cells (7), correlating with our observation that p42 Ebp1 predomi- nantly localizes to the cytoplasm and translocates to the nucleus upon growth factor stimulation (3). Ebp1 represses transcription of both E2F1 (8) and androgen receptor- mediated genes (9, 10). Collectively, these observations sug- gest that p42 Ebp1 acts as a potential tumor suppressor in various cancer cells, whereas p48 Ebp1 may function as an oncogene, promoting cell survival and proliferation. Protein levels of tumor suppressor, p53, largely controlled by MDM2, a ubiquitin E3 ligase (11, 12), are the most important determinants of p53 function. In unstressed conditions, MDM2 promotes the p53 polyubiquitination, leading to proteosome-mediated degradation. Also, Akt- mediated phosphorylation of MDM2 regulates its nuclear translocation, thus stabilizing MDM2 and enhancing MDM2-dependent p53 degradation (13–15). Although the current regulatory model of MDM2 and p53 consists of an autoregulatory feedback loop, and MDM2-mediated p53 degradation depends on high cellular levels of MDM2 (11, 12, 16–18), the mechanisms regulating MDM2-p53 associa- tion are not well understood. It has been suggested that a cofactor may be required for MDM2-mediated p53 degrada- tion, because low levels of MDM2 fail to facilitate polyubi- quitination but rather facilitate monoubiquitination and nuclear export of p53 (17). In addition to interacting with and regulating p53, MDM2 binds to Rb, leading to Rb degradation and activation of E2F1 (19). MDM2 interacts with E2F1, enhancing its stability (20). Although the 2 mRNA transcript expressions are compar- able, in many mammalian cells, p48 is the predominant form Authors' Affiliations: Departments of 1 Molecular Cell Biology and 2 Anat- omy, and 3 Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea; and 4 Department of Anatomy, Seoul National University College of Med- icine; 5 Department of Neurosurgery, Sungkyunkwan University School of Medicine; and 6 Department of Biochemistry and Molecular Biology, Uni- versity of Ulsan, College of Medicine, Seoul, Korea Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). C.K. Kim, T.L.X. Nguyen, and K.M. Joo contributed equally to this work. Corresponding Author: Jee-Yin Ahn, Department of Molecular Cell Biol- ogy, Sungkyunkwan University School of Medicine, 300 Cheoncheon dong, Jangan gu, Suwon 440-746, Korea. Phone: 82-31-299-6134; Fax: 82-31-299-6139; E-mail: jeeahn@skku.edu. doi: 10.1158/0008-5472.CAN-10-1882 Ó2010 American Association for Cancer Research. Cancer Research Cancer Res; 70(23) December 1, 2010 9730 Research. on November 13, 2018. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 23, 2010; DOI: 10.1158/0008-5472.CAN-10-1882