Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin-like growth factor receptor inhibition Ana S. Guerreiro 1 , Danielle Boller 1 , Tarek Shalaby 2 , Michael A. Grotzer 2 and Alexandre Arcaro 1 * 1 Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland 2 Department of Oncology, University Children’s Hospital Zurich, CH-8032 Zurich, Switzerland The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC 50 values ranging from 0.15 to 5 lM. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensi- tivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP- AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combina- tion of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensi- tized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation. ' 2006 Wiley-Liss, Inc. Key words: insulin-like growth factor; neuroblastoma; Akt/protein kinase B Neuroblastoma is the most common extra cranial solid tumor occurring in children and accounts for about 10% of pediatric malig- nancies. 1,2 Current treatments of neuroblastoma such as radiotherapy and chemotherapy are inefficient in subgroups of tumors, because of the resistance of the tumor cells to apoptotic signals. Treatment of high-risk neuroblastoma with prolonged intensive chemotherapy and radiotherapy including peripheral stem cell rescue resulted only in 34% 3-year event-free survival probability. 3 Promising new therapies for neuroblastoma are, however, emerging, which are based on blocking receptor tyrosine kinase (RTK) signaling to some of their downstream signaling targets such as phosphoinositide 3-kinase (PI3K), protein kinase B (PKB)/Akt, the mammalian target of rapamycin (mTOR), the ribo- somal protein S6 kinase (S6K) or mitogen-activated extracellular signal-regulated kinase activating kinase (MEK). Polypeptide growth factors have indeed been shown to play a key role in neu- roblastoma proliferation, chemoresistance and metastasis. IGF sig- naling has been extensively studied in the context of neuroblas- toma proliferation, survival and motility. 4–7 Several potential anti- neuroblastoma therapeutic approaches involving the IGF-I system have been reported. 8 Moreover, inhibition of platelet-derived growth factor receptor (PDGFR) and c-Kit signaling with imatinib mesylate was recently reported to impair growth of a subset of neuroblastoma cell lines. 9 Neurotrophins such as brain-derived neurotrophic factor (BDNF) also play an important role in neuro- blastoma chemoresistance by binding to the Trk receptor family. 10 Fibroblast growth factor-2 (FGF-2), which is expressed in human neuroblastoma cells, may also have chemoprotective and radio- protective effects, given its antiapoptotic functions in other human cancers. 11–13 Finally, an autocrine loop between stem cell factor (SCF) and c-Kit is present in neuroblastoma. 14 Therefore, given that neuroblastoma express a variety of different growth factor receptors, targeting individual receptors may not necessarily pro- vide a successful therapeutic strategy. An alternative approach would be to identify a signaling molecule which lies downstream of several different growth factor receptors and which is essential for transmitting their proliferative and survival message. PI3K is a good example of this, playing a crucial role in control- ling cell proliferation, survival and motility/metastasis down- stream of many different growth factor receptors. 15,16 The impor- tance of PI3K signaling in human cancer is highlighted by the fact that mutations in the tumor suppressor gene PTEN occur fre- quently in human tumors. PTEN is a phosphatase that antagonizes the action of PI3K by dephosphorylating the D-3 position of poly- phosphoinositides. 17 Moreover, recent reports have described acti- vating mutations in the PIK3CA gene encoding the catalytic p110a isoform of PI3K in a variety of human cancers, including breast, colon and ovarian cancer, as well as medulloblastoma. 18 In neuroblastoma, BDNF was shown to protect the tumor cells from chemotherapy-induced apoptosis via the PI3K pathway. 19 IGF-I signaling via PI3K was shown to be required for neuroblastoma differentiation and cytoskeletal rearrangements. 20 Thus, targeting the PI3K/Akt/mTOR/S6K pathway may also represent an attrac- tive novel approach to develop therapies for neuroblastoma. In the present report we have evaluated the potential of the novel IGF-IR inhibitor NVP-AEW541 21 as an antitumor agent in neuroblastoma. Moreover, we have investigated whether targeting downstream signaling mediators of the IGF-IR could enhance the efficacy of the inhibitor in resistant neuroblastoma cell lines. Materials and methods Reagents and antibodies Antibodies against caspase-3, Erk1/2, IGF-IRa and b, IRb, PARP, Akt/PKB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against activated Erk1/2 (Thr202/Tyr204), phosphorylated IGF-IR (Tyr1131), activated Akt/PKB (Ser473) and Ser235/236- and Ser240/244-phosphoryl- ated S6 protein were from Cell Signalling Technology (Danvers, Grant sponsor: Oncosuisse; Grant number: OCS-01501-02-2004; Grant sponsors: Forschungskredit der Universit€ at Z€ urich; StudEx; Fundac ¸~ ao Calouste Gulbenkian. *Correspondence to: Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, Steinwiesstrasse 75, CH-8032 Zur- ich, Switzerland. Fax: 141-1-266-7169. E-mail: alexandre.arcaro@kispi.unizh.ch Received 22 August 2005; Accepted 9 May 2006 DOI 10.1002/ijc.22126 Published online 20 September 2006 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: BDNF, brain-derived neurotrophic factor; EGFR, epider- mal growth factor receptor; Erk, extracellular signal-regulated kinase; FGF-2, fibroblast growth factor-2; IGF-IR, insulin-like growth factor-I re- ceptor; IR, insulin receptor; IRS-1, insulin receptor substrate-1; MEK, mitogen-activated Erk kinase; mTOR, mammalian target of rapamycin; PARP, poly(ADP-ribose) polymerase; PDGFR, platelet-derived growth factor receptor; PDK1, phosphoinositide-dependent kinase-1; PI3K, phos- phoinositide 3-kinase; PKB, protein kinase B; RTK, receptor tyrosine ki- nase; S6K, ribosomal protein S6 kinase; SCF, stem cell factor; SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Int. J. Cancer: 119, 2527–2538 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer