Transactivation of the Epidermal Growth Factor Receptor by Formylpeptide Receptor Exacerbates the Malignant Behavior of Human Glioblastoma Cells Jian Huang, 1 Jinyue Hu, 3 Xiuwu Bian, 4 Keqiang Chen, 1 Wanghua Gong, 2 Nancy M. Dunlop, 1 O.M. Zack Howard, 1 and Ji Ming Wang 1 1 Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research and 2 Basic Research Program, Science Applications International Corporation-Frederick, National Cancer Institute at Frederick, Frederick, Maryland; 3 Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, P.R. China; and 4 Institute of Pathology, South-West Hospital, Third Military Medical University, Chongqing, P.R. China Abstract The G protein-coupled formylpeptide receptor (FPR), which mediates leukocyte migration in response to bacterial and host-derived chemotactic peptides, promotes the chemotaxis, survival, and tumorigenesis of highly malignant human glio- blastoma cells. Because glioblastoma cells may also express other receptors for growth signals, such as the epidermal growth factor (EGF) receptor (EGFR), we investigated the role of EGFR in the signaling cascade of FPR and how two receptors cross-talk to exacerbate tumor growth. We found that N -formyl-methionyl-leucyl-phenylalanine, an FPR agonist peptide, rapidly induced EGFR phosphorylation at tyrosine residue (Tyr) 992, but not residues 846, 1068, or 1173, in glio- blastoma cells, whereas all these residues were phosphor- ylated after only EGF treatment. The FPR agonist-induced EGFR phosphorylation in tumor cells was dependent on the presence of FPR as well as GAi proteins, and was controlled by Src tyrosine kinase. The transactivation of EGFR contributes to the biological function of FPR in glioblastoma cells because inhibition of EGFR phosphorylation significantly reduced FPR agonist-induced tumor cell chemotaxis and proliferation. Fur- thermore, depletion of both FPR and EGFR by short inter- ference RNA abolished the tumorigenesis of the glioblastoma cells. Our study indicates that the glioblastoma-promoting activity of FPR is mediated in part by transactivation of EGFR and the cross-talk between two receptors exacerbates the malignant phenotype of tumor cells. Thus, targeting both receptors may yield antiglioblastoma agents superior to those targeting one of them. [Cancer Res 2007;67(12):5906–13] Introduction Formylpeptide receptor (FPR) is a G protein-coupled receptor (GPCR), originally identified in phagocytic leukocytes, which mediates cell chemotaxis and activation in response to the bacterial chemotactic peptide N -formyl-methionyl-leucyl-phenylal- anine (fMLF). Agonist binding to FPR elicits a cascade of signal transduction pathways that involve phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinases (MAPK), and the transcription factor nuclear factor-nB (for review, see ref. 1). Because of its expression in cells of the immune system and its interaction with bacterial chemotactic peptides, this receptor was thought to participate in host defense against microbial infection. In fact, mice depleted of the FPR analogue FPR1 were more susceptible to infection by Listeria monocytogenes (2). During the past few years, a number of novel and host-derived chemo- tactic agonists of FPR have been identified, including formyl peptides potentially released by mitochondria of ruptured cells (3), Annexin I produced by activated epithelia (4), and a neutrophil granule protein, cathepsin G (5). In addition, functional FPR has been detected in cells of nonhematopoietic origin, such as lung epithelial cells (6) and hepatocytes (7). These findings suggest that FPR may be involved in a broader spectrum of pathophys- iologic processes. Gliomas are the most common tumor type in the brain, characterized by progressive expansion and resistance to conven- tional therapy. The capacity of glioma growth and invasion is closely correlated with the expression of cell surface receptors that sense the signals present in the tumor microenvironment (8, 9). Highly malignant human glioblastoma cells express functional FPR and by responding to potential agonist(s) released by necrotic tumor cells, FPR promotes the directional migration, survival, and production of angiogenic vascular endothelial growth factor (VEGF) by tumor cells (10). Depletion of FPR by short interference RNA (siRNA) markedly reduced the tumorigenicity of the glioblastoma cells in immunodeficient mice (10). In addition, FPR protein was detected in surgical specimens of grade 3 astroblas- toma and grade 4 glioblastoma multiforme in human (10). Thus, both in vitro and in vivo evidence supports the potential role of FPR in promoting the rapid progression of highly malignant human glioma. Human glioma cells may also express the receptor for epidermal growth factor (EGFR; refs. 11–13), which has been implicated as one of the most important growth-stimulating receptors in a great variety of malignant tumors (14) and is a partner of cross-talk with a variety of cell surface receptors. In this study, we investigated the capacity of human glioblastoma cells to concomitantly express both FPR and EGFR and whether these two receptors share intracellular signaling pathways. We found that activation of FPR resulted in transactivation of EGFR in glioblastoma cells and the two receptors interact and synergisti- cally cooperate to exacerbate the malignant behavior of the tumor cells. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Ji Ming Wang, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 31-76, Frederick, MD 21702-1201. Phone: 301-846-6979; Fax: 301-846-7042; E-mail: wangji@mail.ncifcrf.gov. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0691 Cancer Res 2007; 67: (12). June 15, 2007 5906 www.aacrjournals.org Research Article Research. on July 19, 2015. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from