Pathway Contributes to Tumor Cell Survival in Anaplastic Activation of Mammalian Target of Rapamycin Signaling

Anaplastic lymphoma kinase (ALK)–positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosminALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E–binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G1-Sphase regulators, including the cyclin-dependent kinase inhibitors p21 and p27. Apoptosis following inhibition of mTOR expression or function was associated with downregulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKTmediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL. (Cancer Res 2006; 66(13): 6589-97) Introduction Anaplastic large cell lymphoma (ALCL) is an aggressive form of malignant lymphoma of T/null lineage (1). A subset of ALCL tumors carries chromosomal aberrations involving the anaplastic lymphoma kinase (alk) gene on chromosome 2p23 resulting in ALK overexpression (2). The most common of these aberrations is the t(2;5)(p23;q35), which results in expression of the chimeric protein, nucleophosmin-ALK (3). Recent studies have established that nucleophosmin-ALK mediates oncogenesis, at least in part, through phosphorylation/activation of the serine/threonine kinase AKT (4, 5). AKT is a downstream effector of phosphatidylinositol 3-kinase (PI3K) and a critical mediator of mammalian target of rapamycin (mTOR) activity (6). However, the activation status of the mTOR pathway in ALK+ ALCL is unknown. Studying the status of the mTOR pathway in ALK+ ALCL is important because it may provide a rationale for a novel therapeutic strategy for these tumors. mTOR is a serine/threonine protein kinase that forms two distinct protein complexes within cells: one with the regulatoryassociated protein of TOR (raptor) and another with the rapamycin-insensitive companion (rictor; refs. 7, 8). mTOR-raptor complex (composed of mTOR, raptor, and GhL) is sensitive to rapamycin and regulates cell growth, in part, by phosphorylation of the ribosomal protein S6 (rpS6) kinase (p70S6K or S6K1) and subsequent phosphorylation of rpS6 to stimulate protein translation and ribosome biogenesis (9, 10). mTOR-raptor also leads to phosphorylation and inactivation of the eukaryotic initiation factor 4E (eiF4E)–binding protein-1 (4E-BP1), dissociating 4E-BP1 from the RNA cap-binding protein eIF4E, thus promoting cap-dependent translation of mRNA (7, 11, 12). mTOR-rictor complex (composed of mTOR, rictor, and GhL) is not rapamycin sensitive and modulates cell survival and proliferation by direct phosphorylation of AKT on Ser and by facilitating AKT phosphorylation on Thr by PDK1 in vitro (13). Rapamycin is a macrolide antibiotic with antitumor activity (14, 15). Recent in vitro and in vivo studies have shown that rapamycin and its analogues have substantial antitumor activity in hematologic malignancies (16, 17). The mechanism by which rapamycin inhibits mTOR-raptor complex kinase activity is not completely understood. Rapamycin forms an inhibitory complex with the FKBP-rapamycin-binding domain of mTOR, which probably destabilizes the mTOR-raptor interaction and prevents mTOR from phosphorylating p70S6K and 4E-BP1 (18, 19). In this study, we show that the mTOR pathway is frequently activated in ALK+ ALCL cell lines and tumors and that PI3K/AKT contributes to activation of the mTOR pathway in vitro . We also show that inhibition of mTOR expression or activity markedly Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: George Z. Rassidakis, Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Unit 54, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-745-2535; Fax: 713-792-7273; E-mail: [email protected]. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3018 www.aacrjournals.org 6589 Cancer Res 2006; 66: (13). July 1, 2006 Research Article American Association for Cancer Research Copyright © 2006 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-05-3018