Cancer Biology and Signal Transduction Temozolomide Induces the Production of Epidermal Growth Factor to Regulate MDR 1 Expression in Glioblastoma Cells

Glioblastoma multiforme (GBM) commonly resists the frontline chemotherapy treatment temozolomide. The multidrug resistance gene (MDR1) and its protein, P-glycoprotein (P-gp), are associated with chemoresistance. This study investigated the mechanisms underlying MDR1-mediated resistance by GBM to temozolomide. P-gp trafficking was studied by flow cytometry and Western blot analysis. MDR1 expression was analyzed by real-time PCR and reporter gene assays. AP-1 interaction with MDR1 was studied by chromatin immunoprecipitation assay. EGF production was analyzed by ELISA, EGFR signaling was determined by Western blot analysis, and in vivo response to erlotinib and/or temozolomide was studied in nude mice. During the early phase of temozolomide treatment, intracellular P-gp was trafficked to the cell membrane, followedby conformational change into activeP-gp.At the later phase, gene transcription ofMDR1 was inducedby temozolomide-mediatedproduction of EGF.EGFactivatedERK1/2-JNK-AP-1 cofactors (c-jun and c-fos). An inhibitor of EGFR kinase (erlotinib) given to nude mice with GBM prevented temozolomideinduced resistance. The results identified an essential role for activated EGFR in the resistance of GBM to temozolomide. Temozolomide resistance occurred through a biphasic response; first, by a conformational change in P-gp into the active form and, second, by releasing EGF,which caused autocrine stimulation of GBM cells to induce MDR1. Pharmacologic inhibition of EGFR kinase blunted the ability of GBM cells to resist temozolomide. These findingsmay explain reports on the commonoccurrence ofmutant EGFR (EGFRvIII) and EGFR expansion in the resistance of GBM cells. Mol Cancer Ther; 13(10); 2399–411. 2014 AACR. Introduction Resistance to chemotherapeutic agents is a common cause for antineoplastic treatment failure, which leads to oncogenic relapse andmetastasis (1). TheDNAalkylating agent temozolomide is currently the frontline treatment for glioblastoma multiforme (GBM; ref. 2). GBM is a World Health Organization grade IV astrocytoma and the most common adult primary brain tumor. There are several reports that explain temozolomide resistance, such as upregulation of O-6-methylguanine transferase (MGMT; ref. 3). An understanding of the molecular mechanisms underlying temozolomide resistance is essential for the development of combination therapy that could sensitize GBM to temozolomide. The multiple drug resistance-1 (MDR1) gene and its product, P-glycoprotein (P-gp), were the first identified xenobiotic drug ATP-dependent efflux pump (4). Subsequently, >10 other efflux pumps have been identified, collectively referred to as the ATP-binding cassette (ABC) protein family. P-gp is a 170-kDa transmembrane glycoprotein. The MDR1 gene is regulated upstream by a TATAA-less promoter (5). A functional Inr (Initiator) sequence þ1 of the transcriptional initiation site (TIS) has been identified (6). In addition, a number of cis-acting elements have been described, including a GC box, an inverted CCAAT box, and an AP-1 consensus/CCAATlike box (7). The transcriptional activation of the MDR1 gene has been studied in response to different stressors, including chemotherapy (8). In metastatic sarcomas, the MDR1 gene was shown to be induced by doxorubicin, a P-gp target (9). A number of parallels exist between human developmental pathways and oncogenic transformation/maintenance (10). The EGFR signaling pathway represents such overlap.ActivatedEGFRcan form theGrb2/SOS complex to activate the p21 RAS pathway and MAPK/ERK-AP-1 activation (11). EGFR is amplified or mutated in approximately 60% of primary GBM tumors (12). About 40% to 60% of GBMs show constitutive activation of mutant EGFR (EGFRvIII), resulting in poor clinical prognosis (13). The EGFR has tyrosine kinase functions and serves New Jersey Medical School, Rutgers University, Newark, New Jersey. Department of Chemistry, University of Puerto Rico-Cayey, Cayey, Puerto Rico. Robert Wood Johnson Medical School, Cancer Institute of New Jersey, Rutgers University, New Brunswick, New Jersey. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). CorrespondingAuthor:Pranela Rameshwar, NewJerseyMedical School, Rutgers School of Biomedical Health Sciences, 185 SouthOrange Avenue, MSB E-585, Newark, NJ 07103. Phone: 973-972-0625; Fax: 973-9728854; E-mail: rameshwa@njms.rutgers. edu doi: 10.1158/1535-7163.MCT-14-0011 2014 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals. org 2399 on June 24, 2017. © 2014 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0011