Mammalian Target of Rapamycin Inhibition Promotes Response to Epidermal Growth Factor Receptor Kinase Inhibitors in PTEN-Deficient and PTEN-Intact Glioblastoma Cells Maria Y. Wang, 1,8 Kan V. Lu, 1 Shaojun Zhu, 1 Ederlyn Q. Dia, 1 Igor Vivanco, 4 Gregory M. Shackleford, 7 Webster K. Cavenee, 9 Ingo K. Mellinghoff, 4 Timothy F. Cloughesy, 2,5 Charles L. Sawyers, 3,4,6 and Paul S. Mischel 1,5 Departments of 1 Pathology and Laboratory Medicine, 2 Neurology, 3 Medicine, 4 Molecular and Medical Pharmacology, 5 Henry E. Singleton Brain Tumor Program, and 6 Howard Hughes Medical Institute, David Geffen School of Medicine, University of California at Los Angeles; 7 Departments of Pediatrics, and Molecular Microbiology and Immunology, Keck School of Medicine at University of Southern California, Los Angeles, California; 8 Ventana Medical Systems, Tucson, Arizona; and 9 Ludwig Institute for Cancer Research at University of California, San Diego, California Abstract The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma, making it a compelling molecular target for therapy. We have recently shown that coexpression of EGFRvIII and PTEN protein by glioblastoma cells is strongly associated with clinical response to EGFR kinase inhibitor therapy. PTEN loss, by dissociating inhibition of the EGFR from downstream phosphatidylinositol 3-kinase (PI3K) pathway inhibition, seems to act as a resistance factor. Because 40% to 50% of glioblastomas are PTEN deficient, a critical challenge is to identify strategies that promote responsiveness to EGFR kinase inhibitors in patients whose tumors lack PTEN. Here, we show that the mammalian target of rapamycin (mTOR) inhibitor rapamycin enhances the sensitivity of PTEN- deficient tumor cells to the EGFR kinase inhibitor erlotinib. In two isogenic model systems (U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN in relevant combina- tions, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored), we show that combined EGFR/mTOR kinase inhibition inhibits tumor cell growth and has an additive effect on inhibiting downstream PI3K pathway signaling. We also show that combination therapy provides added benefit in promoting cell death in PTEN-deficient tumor cells. These studies provide strong rationale for combined mTOR/EGFR kinase inhibitor therapy in glioblas- toma patients, particularly those with PTEN-deficient tumors. (Cancer Res 2006; 66(16): 7864-9) Introduction In cancer, key regulators of intracellular signaling, including receptor tyrosine kinases, are commonly overactive. These persistent signals promote tumor growth and invasion, but may also provide attractive targets for therapy (1). In glioblastoma, the most common malignant primary brain tumor of adults, the epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase is commonly amplified (2, 3). This amplification often occurs in association with expression of a persistently active mutant receptor (EGFRvIII; ref. 3). In early clinical trials, 10% to 20% of malignant glioma patients seem to derive benefit from the EGFR kinase inhibitors erlotinib and gefitinib (4, 5). We have recently shown that coexpression of the EGFRvIII oncogene and the PTEN tumor suppressor protein is strongly associated with clinical response to EGFR kinase inhibitor therapy in two independent sets of malignant glioma patients (6). We also showed that EGFRvIII sensitizes glioblastoma cells to EGFR kinase inhibitors, whereas PTEN loss confers resistance to EGFR tyrosine kinase inhibitors in isogenic cell systems (6). Because glioblastomas frequently lose or have altered PTEN tumor suppressor activity (2, 7, 8), developing strategies to promote clinical response to EGFR kinase inhibitors in these patients is a critical challenge. PTEN loss promotes resistance to EGFR kinase inhibitors in part by dissociating EGFR inhibition from downstream phosphatidyli- nositol 3-kinase (PI3K) pathway inhibition (6, 9–11). Therefore, we hypothesized that targeting the PI3K/Akt signaling pathway downstream of PTEN could enhance the sensitivity of PTEN- deficient glioblastoma cells to EGFR kinase inhibitors. Here, we use two isogenic model systems—U87MG glioblastoma cells expressing EGFR, EGFRvIII, and PTEN proteins in relevant combinations, and SF295 glioblastoma cells in which PTEN protein expression has been stably restored—to examine the effect of mammalian target of rapamycin (mTOR) kinase inhibition in promoting response of PTEN-deficient tumor cells to erlotinib. Materials and Methods Cell lines and reagents. The human glioblastoma cell line U87MG was purchased from American Type Culture Collection (Rockville, MD), and human SF295 glioblastoma cells were from the NCI-60 cell line panel. All cell lines were routinely maintained in DMEM containing 10% fetal bovine serum. Erlotinib (Tarceva, OSI-774) was kindly provided by Genentech, Inc. (South San Francisco, CA). Rapamycin and human EGF were purchased from Sigma (St. Louis, MO). Gene construction and retroviral infection. The full-length human PTEN cDNA was cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA), transfected into U87MG and SF295 cells, and single clones resistant to geneticin selection were isolated to generate the stable cell lines U87MG-PTEN and SF295-PTEN. Human EGFR and EGFRvIII cDNAs were PCR amplified from pLWERNL and pLERNL plasmids and cloned into the retroviral expression vectors Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M.Y. Wang and K.V. Lu contributed equally to this work. C.L. Sawyers is an Investigator of the Howard Hughes Medical Institute and is a Doris Duke Distinguished Clinical Investigator. Requests for reprints: Paul S. Mischel, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte, Los Angeles, CA 90095-1732. Phone: 310-794-5223; Fax: 310-206-8290; E-mail: pmischel@mednet.ucla.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-4392 Cancer Res 2006; 66: (16). August 15, 2006 7864 www.aacrjournals.org Priority Report