Expression of TIP-1 Confers Radioresistance of Malignant Glioma Cells Miaojun Han1,3,4, Hailun Wang 1 , Hua-Tang Zhang 3 , Zhaozhong Han1,2,5* 1 Department of Radiation Oncology, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Cancer Biology, Vanderbilt University, Nashville, Tennessee, United States of America, 3 Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan Province, China, 4 Graduate School, Chinese Academy of Sciences, Beijing, China, 5 Vanderbilt-Ingram Cancer Center, School of Medicine, Vanderbilt University, Nashville, Tennessee, United States of America Abstract Background: Malignant gliomas represent one group of tumors that poorly respond to ionizing radiation (IR) alone or combined with chemotherapeutic agents because of the intrinsic or acquired resistance. In this study, TIP-1 was identified as one novel protein that confers resistance of glioma cells to IR. Methodology/Principal Findings: Meta-analysis indicated that high TIP-1 expression levels correlate with the poor prognosis of human malignant gliomas after radiotherapy. Studies with established human glioma cell lines demonstrated that TIP-1 depletion with specific shRNAs sensitized the cells to IR, whereas an ectopic expression of TIP-1 protected the glioma cells from the IR-induced DNA damage and cell death. Biochemical studies indicated that TIP-1 protein promoted p53 ubiquitination and resulted in a reduced p53 protein level. Furthermore, p53 and its ubiquitination are required for the TIP-1 regulated cellular response to IR. A yeast two-hybrid screening identified that TIP-1, through its single PDZ domain, binds to the carboxyl terminus of LZAP that has been studied as one tumor suppressor functioning through ARF binding and p53 activation. It was revealed that the presence of TIP-1 enhances the protein association between LZAP and ARF and modulates the functionality of ARF/HDM2 toward multi-ubiquitination of p53, while depleting TIP-1 rescued p53 from polyubiquitination and degradation in the irradiated glioma cells. Studies with a mouse xenograft model indicated that depleting TIP-1 within D54 cells improved the tumor growth control with IR. Conclusions/Significance: This study provided the first evidence showing that TIP-1 modulates p53 protein stability and is involved in the radioresistance of malignant gliomas, suggesting that antagonizing TIP-1 might be one novel approach to sensitize malignant gliomas to radiotherapy. Citation: Han M, Wang H, Zhang H-T, Han Z (2012) Expression of TIP-1 Confers Radioresistance of Malignant Glioma Cells. PLoS ONE 7(9): e45402. doi:10.1371/ journal.pone.0045402 Editor: Michel M. Ouellette, University of Nebraska Medical Center, United States of America Received February 29, 2012; Accepted August 22, 2012; Published September 17, 2012 Copyright: 2012 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by National Institutes of Health (NIH) grants R01CA127482 (ZH) and P50 CA128323 (J. Gore). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Zhaozhong.han@vanderbilt.edu Introduction Malignant gliomas are the most common malignant primary tumors arising in the human brain, treating these life-threatening tumors is still a clinical challenge. Although most of the glioma patients are treated with radiotherapy alone or combined with surgical resection and/or chemotherapies, outcomes of the treatment courses are still unsatisfactory partially due to either intrinsic or acquired tumor resistance to the cytotoxic treatment [1,2]. The tumor suppressor p53 protein plays a vital role in the cellular responses to cytotoxic treatments through regulating cell cycle progression, DNA damage repair, and cell death. Upon stimulation of intrinsic or extrinsic factors, p53 protein is activated to sense, transduce and execute cellular responses. Human double minute 2 (HDM2) (or the murine homologue, MDM2 in mouse) has been well characterized as a negative regulator of p53 through two main mechanisms: direct binding to the N-terminal end of p53 protein and inhibiting p53-regulated transcription [3,4], or targeting p53 for ubiquitination and subsequent degradation through the 26S proteasome [5]. HDM2, in turn, is regulated by p14/p19 ARF (alternative reading frame) which binds to HDM2 and limits the HDM2-regulated p53 inhibition [6]. Dysfunctional mutations in the ARF/HDM2/p53 pathway, such as over activation of HDM2 or a loss-of-function mutation of p53, have been demonstrated as mechanisms by which tumor cells acquire the unlimited proliferation potentials and resistance to apoptosis in a broad spectrum of tumors [7,8]. In fact, restoring the functionality of p53 protein has been proposed and tested for clinical treatment of cancers [7,9]. However, in spite of the fact that a significant portion of the primary malignant gliomas express the wild-type p53 protein, the status of p53 expression has a poor prognostic value in predicting the therapeutic outcomes of the primary malignant gliomas [10,11]. Therefore, more studies are needed to understand the complexity of p53 activation and functionality in tumor progression and response to therapeutics. Recently, LZAP (also known as CDK5 regulatory subunit- associated protein 3 or C53) has been characterized as a tumor PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45402