RIP 1 Activates PI 3 K-Akt via a Dual Mechanism Involving NFK B – Mediated Inhibition of the mTOR-S 6 KIRS 1 Negative Feedback Loop and Down-regulation of PTEN

Therapeutic inhibition of mammalian target of rapamycin (mTOR) in cancer is complicated by the existence of a negative feedback loop linking mTOR to the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Thus, mTOR inhibition by rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP1) plays a key role in nuclear factor-KB (NFKB) activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma multiforme, the most common adult primary malignant brain tumor, but not in grade II to III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. First, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals that regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-KB– dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is down-regulation of cellular PTEN levels, which appears to be independent of NF-KB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in glioblastoma multiforme. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-KB pathway. Our data suggest that the RIP1NF-KB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis. [Cancer Res 2009;69(10):4107–11] Introduction Activation of mammalian target of rapamycin (mTOR) may play an important role in cancer (1). The phosphatidylinositol 3kinase (PI3K)-Akt signaling pathway plays an essential role in the activation of mTOR. PTEN is a lipid phosphatase that is commonly mutated in human cancer and is the key negative regulator of PI3K-Akt activity upstream of Akt (2). mTOR is also involved in a negative feedback regulation of PI3K-Akt (3). Thus, exposure of cells to rapamycin or silencing mTOR with small interfering RNA (siRNA) leads to PI3K-Akt activation (3, 4). An important mechanism mediating this negative feedback loop is via p70S6K-IRS1 (3, 5). Increased activation of mTOR results in activation of p70S6K, which phosphorylates IRS1 resulting in inhibition of IRS1. The IGF/insulin and IRS1 signaling pathways are major activators of the PI3K-Akt pathway. Thus, mTORp70S6K-mediated inhibition of IRS1 results in decreased PI3K-Akt activation. Aberrant activation of the PI3K-Akt-mTOR axis is well documented in cancer and has led to efforts to target mTOR. This feedback loop may explain the limited efficacy of mTOR inhibition in certain cancers (6), because increased activation of PI3K-Akt could activate mTOR-independent oncogenic signaling pathways downstream of Akt. The receptor interacting protein (RIP1) is involved in the activation of two key prosurvival pathways, the nuclear factor-nB (NF-nB; refs. 7, 8) and PI3K-Akt pathways (9, 10). The mechanism used by RIP1 to activate PI3K-Akt is unknown. RIP1 is widely expressed and is a member of a kinase family that mediates cellular response to inflammation and stress (7, 11, 12). RIP1 is expressed both constitutively and inducibly in response to inflammatory cytokines (7, 11, 13–15). We have recently shown that RIP1 levels are increased in f30% of patients with glioblastoma multiforme, the most common primary brain tumor in adults, and confers a worse prognosis in this disease (16). In this study, we show that RIP1 negatively activated PI3K-Akt by regulating two key inhibitors of the PI3K-Akt pathway, upstream and downstream of Akt, and show that RIP1 links NF-nB activation to the mTOR-PI3K-Akt negative feedback loop. Materials and Methods Antibodies. InBa antibodies were obtained from Santa Cruz Biotechnology; RIP antibody was from BD Biosciences for both Western blot and immunohistochemistry and FLAG, mTOR, and pAkt S473 for both Western blot and immunohistochemistry; PTEN, pp70S6K Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Park and D. Zhao contributed equally to this work. Current address for S. Park: Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX. Requests for reprints: Amyn A. Habib, University of Texas Southwestern Medical Center, Mail Code 8813, 6001 Forest Park ND4.136, Dallas, TX 75390-8813. Phone: 214-645-6237; Fax: 214-645-6240; E-mail: Amyn.Habib@UTSouthwestern.edu. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-0474 www.aacrjournals.org 4107 Cancer Res 2009; 69: (10). May 15, 2009 Priority Report