[CANCER RESEARCH 63, 364 –374, January 15, 2003] Insulin-like Growth Factor I-mediated Protection from Rapamycin-induced Apoptosis Is Independent of Ras-Erk1-Erk2 and Phosphatidylinositol 3-Kinase-Akt Signaling Pathways 1 Kuntebommanahalli N. Thimmaiah, John Easton, Shile Huang, Karen A. Veverka, Glen S. Germain, Franklin C. Harwood, and Peter J. Houghton 2 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794 ABSTRACT The mTOR inhibitor rapamycin induces G 1 cell cycle accumulation and p53-independent apoptosis of the human rhabdomyosarcoma cell line Rh1. Insulin-like growth factor I (IGF-I) and insulin, but not epidermal growth factor or platelet-derived growth factor, completely prevented apoptosis of this cell line. Because the Ras-Erk1-Erk2 and phosphatidy- linositol 3-kinase (PI3K)-Akt pathways are implicated in the survival of various cancer cells, we determined whether protection from rapamycin- induced apoptosis by IGF-I requires one or both of these pathways. Despite the blocking of Ras-Erk signaling by the addition of PD 98059 (a MEK1 inhibitor) or by the overexpression of dominant-negative RasN17, IGF-I completely prevented rapamycin-induced death. Inhibition of Ras signaling did not prevent Akt activation by IGF-I. To determine the role of the PI3K-Akt pathway in rescuing cells from apoptosis caused by rapamycin, cells expressing dominant-negative Akt were tested. This mutant protein inhibited IGF-I-induced phosphorylation of Akt and blocked phosphorylation of glycogen synthase kinase 3. The prevention of rapamycin-induced apoptosis by IGF-I was not inhibited by expression of dominant-negative Akt either alone or under conditions in which LY 294002 inhibited PI3K signaling. Furthermore, IGF-I prevented rapamycin-induced apoptosis when the Ras-Erk1-Erk2 and PI3K-Akt pathways were blocked simultaneously. Similar experiments in a second rhabdomyosarcoma cell line, Rh30, using pharmacological inhibitors of PI3K or MEK1, alone or in combination, failed to block IGF-I rescue from rapamycin-induced apoptosis. Therefore, we conclude that a novel pathway(s) is responsible for the IGF-I-mediated protection against ra- pamycin-induced apoptosis in these rhabdomyosarcoma cells. INTRODUCTION IGFs 3 , IGF-I and IGF-II are soluble peptide factors that circulate while bound to one of six IGF-binding proteins. The IGFs, their receptors, and the IGF-binding proteins constitute a family of cellular modulators that play essential roles in regulating growth and devel- opment (1). The action of IGFs results primarily from the activation of the IGF-IR (2). This receptor resembles the insulin receptor in structural as well as functional aspects (3), and is a heterotetrameric transmembrane glycoprotein consisting of 2 - and 2 -subunits. The tyrosine kinase catalytic site and the ATP-binding site are located in the cytoplasmic portion of the -subunit. The tyrosine kinase activity of the -subunit is stimulated when IGF-I binds to the -subunit. Intracellular substrates of the IGF-IR include the insulin receptor substrates IRS-1 and IRS-2, and the SH2-containing protein Shc. Phosphorylation of these proteins results in their interaction with other signaling molecules, such as the adapter Grb2, that are, in turn, coupled to effector molecules, such as the guanine nucleotide ex- change factor Sos (4 – 6) and PI3K (7, 8). The activated Shc-Grb2-Sos complex activates the small GTP-binding protein Ras, and activated Ras initiates a cascade of Ser/Thr kinases, some of which eventually translocate to the nucleus to stimulate gene expression (9, 10). The extracellular signal-regulated MAPKs, Erk1 and Erk2, are key inter- mediates in the propagation of signals from many growth factor receptors to the nucleus (9, 11). Erk proteins are activated by MEK, a dual function kinase that phosphorylates tyrosine and threonine residues of Erk (12, 13) downstream of Ras and Raf1. IRS-1 and IRS-2 lie upstream of a signaling cascade involving PI3K. Phosphoinositides phosphorylated by PI3K recruit PDKs such as PDK1 to the plasma membrane. Evidence suggests that phospho- rylation of Thr308 and Ser473 of Akt by PDK1 and PDK2 activates Akt (14 –16). This event results in Akt phosphorylation of its down- stream substrates such as GSK-3 (17), 6-phosphofructo-2-kinase (18), the Bcl2 family member Bad (19), caspase-9 (20), nitric oxide syn- thase (21, 22), and the winged-helix family of FKHRL1 transcription factors (23). Activation of these substrates leads to glucose transport, glycolysis, glycogen synthesis, and cell survival (24, 25). Thus, IGF- IR, when activated by its ligands, plays an important role in the growth of cells by inducing mitogenesis and transformation, and by protecting cells from various apoptotic injuries (26). Several reports have documented recently the involvement of IGF-I in regulating apoptosis (27) induced by various stimuli, including physiological stress (28), hyperosmosis (29, 30), chemotherapy (31), and DNA damage caused by chemotherapeutic drugs or UV-B radiation (32–34). Additional evidence of the role of IGF-I in regulating apoptosis has been provided by studies involving rapamycin, an immunosuppressive macrocyclic lactone that specifically inhibits the activity of mTOR, a Ser/Thr kinase downstream of PI3K. Inhibition of mTOR leads to G 1 arrest of many malignant cell lines, and currently analogs of rapamy- cin are being investigated as cancer therapeutic agents. We have reported previously that rapamycin selectively induces apoptosis of tumor cells that express mutant p53 and are grown under serum-free conditions (35). However, the addition of IGF-I to the growth medium completely protects Rh1 cells from rapamycin-induced apoptosis. Therefore, we are interested in understanding how IGF-I protects Rh1 cells from apoptosis induced by rapamycin. Receptor tyrosine kinases such as those for the receptors of IGF-I, insulin, and PDGF stimulate nuclear events by activating cascades of protein kinases (4). EGF activates the PI3K-Akt signaling pathway in several EGF receptor-overexpressing cells such as prostate cancer cells (36), epidermoid cancer cells (37), and ovarian cancer cells (38). Received 4/17/02; accepted 11/13/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by USPHS awards CA77776, CA23099, and CA28765 (Cancer Center Support Grant) from the National Cancer Institute; by a grant from Wyeth-Ayerst Laboratories; and by the American Lebanese Syrian Associated Charities (ALSAC). 2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Mail Stop 230, 332 North Lauder- dale Street, Memphis, TN 38105-2794. Phone: (901) 495-3440; Fax: (901) 495-4290; E-mail: peter.houghton@stjude.org. 3 The abbreviations used are: IGF-I, insulin-like growth factor; ERK, extracellular signal-regulated kinase; RBD, Ras-binding domain; GFP, green fluorescent protein; RIPA, radioimmunoprecipitation assay; PDK, phosphoinositide-dependent kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; IGF-IR, insulin- like growth factor I receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphati- dylinositol 3'-kinase; EGF, epidermal growth factor; PDGF; platelet-derived growth factor; GSK-3, glycogen synthase kinase 3; MN2E, modified N2E medium; FACS, fluorescence-activated cell sorting; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase. 364 Research. on September 9, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from