Research Article The Receptor Interacting Protein 1 Inhibits p53 Induction through NF-KB Activation and Confers a Worse Prognosis in Glioblastoma Seongmi Park,1 Kimmo J. Hatanpaa,2,7 Yang Xie,3,8 Bruce E. Mickey,4,7 Christopher J. Madden,4,7 Jack M. Raisanen,2,7 Deepti B. Ramnarain,1 Guanghua Xiao,3 Debabrata Saha,5 David A. Boothman,8 Dawen Zhao,6 Robert M. Bachoo,1,7,8 Russell O. Pieper,9 and Amyn A. Habib1,7,8 Departments of 1Neurology, 2Pathology, 3Clinical Sciences, 4Neurosurgery, 5Radiation Oncology, and 6Radiology, 7Annette G. Strauss Center of Neurooncology, and 8Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas and 9Department of Neurological Surgery, University of California-San Francisco, San Francisco, California Abstract studies have linked components of the NF-nB signaling pathway to Nuclear factor-KB (NF-KB) activation may play an important cell cycle progression and tumorigenesis (11–16). role in the pathogenesis of cancer and also in resistance to An intriguing mechanism underlying the pathogenesis of treatment. Inactivation of the p53 tumor suppressor is a key inflammation-induced cancer is the negative regulation of tumor component of the multistep evolution of most cancers. Links suppressor pathways by inflammatory and stress-induced signals. between the NF-KB and p53 pathways are under intense p53 is a key tumor suppressor altered in a broad range of human investigation. In this study, we show that the receptor cancers, including glioma, and an important outcome of p53 interacting protein 1 (RIP1), a central component of the activation is cell cycle arrest or apoptosis after DNA damage K (17, 18). Previous studies have documented links between the NF- NF- B signaling network, negatively regulates p53 tumor sup- n pressor signaling. Loss of RIP1 from cells results in augmented B and p53 networks that have largely been reported to be induction of p53 in response to DNA damage, whereas antagonistic (19) but may also be synergistic (20). There is evidence that components of the NF-nB signaling network interact with p53 increased RIP1 level leads to a complete shutdown of DNA h damage–induced p53 induction by enhancing levels of cellular at multiple levels. For example, IKK2 (IKK ) inhibits p53 induction in response to chemotherapeutic drugs via an up-regulation of mdm2. The key signal generated by RIP1 to up-regulate mdm2 a and inhibit p53 is activation of NF-KB. The clinical implication mdm2 (21), whereas IKK1 (IKK ) interferes with p53-mediated gene transcription by inducing CBP phosphorylation (22). Thus, of this finding is shown in glioblastoma, the most common n primary malignant brain tumor in adults. We show that RIP1 NF- B pathway–mediated inhibition of p53 function may promote is commonly overexpressed in glioblastoma, but not in grades the pathogenesis of cancer. II and III glioma, and increased expression of RIP1 confers a The death domain–containing kinase receptor interacting worse prognosis in glioblastoma. Importantly, RIP1 levels protein 1 (RIP1, RIPK1) is an essential component of the signaling n correlate strongly with mdm2 levels in glioblastoma. Our cascade that activates NF- B in response to cellular stress and results show a key interaction between the NF-KB and p53 inflammation (23, 24). Thus, RIP1 is required for tumor necrosis a a n pathways that may have implications for the targeted factor (TNF )–induced and DNA damage–induced NF- B treatment of glioblastoma. [Cancer Res 2009;69(7):2809–16] activation (25–28). In addition, RIP1 is also a key component of innate immunity, essential for TLR-3–mediated activation of NF-nB (29). RIP1 is composed of kinase, intermediate, and death domains. Introduction RIP1 is involved in the activation of the IKKs via a kinase- A link between chronic inflammation and cancer has been independent mechanism (30). Thus, RIP1 seems to function as an suspected for over a century (1). A major link between adaptor, and it is the intermediate domain of RIP1 that is essential n inflammation and cancer is mediated by activation of nuclear for NF- B activation. factor-nB(NF-nB; refs. 2–4). NF-nB activation is a strong Glioblastoma (glioblastoma multiforme) is the most common prosurvival signal (5). Constitutive and deregulated activation of primary malignant brain tumor in adults and is resistant to NF-nB is widespread in human cancer (3, 6) and promotes survival treatment (31). The median survival of glioblastoma patients with of tumor cells and resistance to treatment (7, 8). Furthermore, radiation and chemotherapy was recently noted to be 14.6months experimental models support a causal role for NF-nB activation in (32). The molecular pathogenesis of glioblastoma includes genetic inflammation-induced cancer (9, 10). Cross-talk between stress- alterations in pathways mediating proliferation, apoptosis, and cell induced/inflammatory responses and oncogenic signaling path- cycle control (33, 34). Inflammatory responses can be readily ways is likely to play an important role in cancer. A number of detected in glioma in the form of infiltrating macrophages/ microglia and lymphocytes, production of inflammatory cytokines, and activation of NF-nB (35, 36). Importantly, NF-nB activation Note: Supplementary data for this article are available at Cancer Research Online may be linked to the resistance of glioblastoma cells to (http://cancerres.aacrjournals.org/). O6-alkylating agents (37, 38). S. Park and K.J. Hatanpaa contributed equally to this work. Requests for reprints: Amyn A. Habib, University of Texas Southwestern Medical In this study, we show that RIP1 negatively regulates p53 Center, Mail Code 8813, 6001 Forest Park ND4.136, Dallas, TX 75390-8813. Phone: 214- induction in response to DNA damage. RIP1 regulates p53 via the 645-6237; Fax: 214-645-6240; E-mail: [email protected]. I2009 American Association for Cancer Research. up-regulation of mdm2 levels. We also elucidate a key role for doi:10.1158/0008-5472.CAN-08-4079 NF-nB activation in RIP1-mediated regulation of mdm2 and p53 www.aacrjournals.org 2809 Cancer Res 2009; 69: (7). April 1, 2009 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Cancer Research down-regulation. Analysis of glioma patient samples showed that low groups based on a RIP1 value of 1.2, which represents a 50% increase RIP1 is overexpressed in f30% of glioblastoma (grade IV), but not in over the levels found in normal brain. To investigate whether RIP1 level grades II and III glioma. Importantly, there was a striking correlation (ratio between RIP1 and ERK2) is associated with the clinical outcome, we between RIP1 and mdm2 levels in glioblastoma, consistent with our applied a proportional hazard model using both overall survival time and progression-free survival time as the outcomes (censored by the last known mechanistic data in vitro. Finally, high RIP1 level is an independent number of follow-up months). The multivariate proportional hazard model negative prognostic indicator in glioblastoma. was used to analyze the effect of additional prognosis factors, such as age and Karnofsky scores, with the RIP1 level. The survival curves were fitted using the Kaplan-Meier method. The log-rank test was used to compare Materials and Methods different survival curves. Fisher’s exact test was used to calculate the P value Plasmids and cell lines. RIP1 plasmid was obtained from Dr. Brian Seed for association between RIP1 and mdm2 in glioblastoma. The survival and cloned into pcDNA3.1 with a COOH terminal FLAG tag using standard analysis was performed using the survival package in SAS software. +/+ À/À molecular techniques. rip1 and rip1 murine embryo fibroblasts were provided by Dr. Michelle Kelliher (27). In this study, we used primary mouse embryo fibroblasts (MEF) at passages 3 to 4. A p21-LUC promoter was Results provided by Dr. Bert Vogelstein. RIP1 influences p53 induction in response to DNA damage. Antibodies, reagents, and Western blotting. p53 (DO1), InBa, p65, and Previous studies have documented interactions between compo- extracellular signal-regulated kinase 2 (ERK2) antibodies were obtained n from Santa Cruz Biotechnology. The RIP antibody was purchased from BD nents of the NF- B signaling network and p53. Thus, we Biosciences and was used for both immunohistochemistry and Western investigated whether the cellular level of RIP1 could influence À/À blotting. mdm2 (2A10) antibodies were purchased from Calbiochem. p53 p53 induction in response to DNA damage. Exposure of RIP1 (1C12), p21, and FLAG were purchased from Cell Signaling Technology. We primary MEFs to ionizing radiation (IR) resulted in a significantly used p53 DO1 antibodies for the detection of p53 in (human) glioma cell greater induction of p53 compared with wild-type cells (Fig. 1A). À À lines, whereas p53 1C12 antibodies were used for the detection of (mouse) Similarly, exposure of RIP1 / MEFs to UV radiation elicited a p53 in MEFs. For tumor samples, 30 Ag of protein were loaded on to 7.5% greater induction of p53 (Supplementary Fig. S1A). Next, we polyacrylamide gels, and Western blotting was performed according to investigated the effect of increased RIP1 expression in the human standard protocols. glioblastoma cell line U87MG, which expresses wild-type p53. To Luciferase assays. U87MG cells (70K) were plated in 24-well dishes overexpress RIP1, we used a tetracycline-inducible adenoviral followed by transfection with either p21-LUC along with RIP plasmid or empty vector, using calcium phosphate. A dual-luciferase reporter assay expression vector (tet-off). An empty adenoviral vector (Ad-null) C system was used according to the instructions of the manufacturer was used as an additional control (Supplementary Fig. S1 ). Cells (Promega). Firefly luciferase activity was measured in a luminometer and were exposed to IR (10 Gy) and then followed by Western blot. normalized on the basis of Renilla luciferase activity.