[CANCER RESEARCH 63, 5629 –5635, September 1, 2003] Epidermal Growth Factor Receptor-stimulated Activation of Phospholipase C-1 Promotes Invasion of Head and Neck Squamous Cell Carcinoma 1 Sufi Mary Thomas, Francesca M. Coppelli, Alan Wells, William E. Gooding, John Song, Jareer Kassis, Stephanie D. Drenning, and Jennifer Rubin Grandis 2 Departments of Otolaryngology [S. M. T., F. M. C., J. S., S. D. D., J. R. G.], Pharmacology [J. R. G.], Pathology [A. W., J. K.], and Biostatistics [W. E. G.], University of Pittsburgh, University of Pittsburgh Cancer Institute, and Pittsburgh Veterans’ Administration Medical Center [A. W., J. K.], Pittsburgh, Pennsylvania 15213 ABSTRACT Lymph node metastasis and local invasion of head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. However, little is known about the factors governing tumor cell invasion in HNSCC. Phospholipase C-1 (PLC-1) contributes to tumor cell invasion in ex- perimental systems when activated by the epidermal growth factor recep- tor (EGFR). We hypothesized that EGFR overexpression in HNSCC mediates invasion via PLC-1. On EGFR ligand stimulation, phosphoryl- ation of PLC-1 increased in all of the HNSCC cell lines tested (4 of 4). In the presence of EGFR-specific tyrosine kinase inhibitor (PD153035) or an anti-EGFR antibody (C225), PLC-1 activation was abrogated indicating that PLC-1 was downstream of EGFR. Blocking cellular PLC with an inhibitor (U73122) reduced inositol phosphate turnover in all of the HNSCC cell lines examined, and treatment with the PLC inhibitor or antisense oligonucleotides targeting PLC-1 significantly reduced in vitro invasiveness of HNSCC cell lines through Matrigel. To determine the clinical relevance of these findings, we compared levels of PLC-1 in tumor and paired normal tissue from 33 patients with HNSCC. PLC-1 levels were significantly higher (P < 0.0001) in the tumors compared with the normal mucosa of HNSCC patients. Levels of activated PLC-1 were analyzed in 20 patients. Tumors expressed higher levels of phosphorylated PLC-1 compared with normal adjacent mucosa (P  0.05). Thus, PLC-1 may mediate invasion and metastasis downstream of EGFR in HNSCC. INTRODUCTION Nearly 50% of patients with HNSCC 3 present with cervical lymph node metastases (1). Invasion of the tumor into the neck viscera is a primary cause of morbidity and mortality in this cancer. The mecha- nisms that govern tumor cell invasion in HNSCC are incompletely understood. Elucidation of the molecular events that mediate invasion is required to improve therapeutic approaches and, hence, survival. More than 95% of HNSCC tumors express elevated levels of the EGFR (2, 3). We have shown previously that increased EGFR ex- pression in HNSCC tumors correlates with reduced survival and an increased incidence of lymph node metastasis (4). An established oncogene, EGFR mediates cellular motility, proliferation, and pre- vents apoptosis in HNSCC cells via activation of a number of down- stream signaling pathways. Activation of these pathways by EGFR is necessary for tumor progression (5). However, to rationally disrupt these events, an increased understanding of EGFR signaling is re- quired to elucidate its biological role in cancers including HNSCC. Several signaling pathways downstream of EGFR have been re- ported in HNSCC including the MAPK, phosphatidylinositol 3'- kinase, and STATs (6). Several lines of evidence suggest a redun- dancy among EGFR signaling pathways (7), whereas others have reported modulation of a specific phenotype when one pathway is specifically targeted. In HNSCC cells, EGFR-stimulated MAPK ac- tivation induced proliferation but not invasion (8). The phosphatidy- linositol 3'-kinase pathway has been implicated in mediating anti- apoptotic functions and conferring radiation-resistance in HNSCC cells (9). We have shown previously that EGFR-mediated STAT3 activation is required for cell growth and survival in vitro (10). Additional evidence suggests that constitutively activated STAT3 in HNSCC tumors results in uncontrolled cell growth by an antiapoptotic mechanism (11). Phosphoinositide bisphosphate turnover downstream from PLC activation has not been studied previously in HNSCC. The present study was undertaken to test the hypothesis that EGFR stimulation of PLC-1 mediates cell invasion in HNSCC. We exam- ined PLC-1 expression and activation in a series of HNSCC cell lines and patient tissues. Blockade of EGFR abrogated PLC-1 levels suggesting that PLC-1 activation in HNSCC cells was primarily because of EGFR stimulation. Abrogation of PLC-1 decreased HNSCC cell invasion in vitro without affecting cell proliferation. In vivo, PLC-1 was expressed and phosphorylated at higher levels in tumor tissue compared with normal adjacent mucosa. These results indicate that EGFR-mediated PLC-1 activation modulates invasion of HNSCC cells and may contribute to tumorigenesis. MATERIALS AND METHODS Cell Lines and Tissues. Cell lines derived from HNSCC were maintained in DMEM with 10% FBS (Life Technologies, Inc., Grand Island, NY). The OSC-19 cell line was cultured in Eagle’s MEM containing 10% FBS and nonessential amino acids (0.1 mM). Cell lines OSC-19 (12) and PCI-15b were derived from metastatic lymph nodes (13). UM-22a was derived from a SCC of the buccal mucosal (14). UPCI:SCC32, derived from the retromolar trigone, is a generous gift from Dr. Susanne M. Gollin (University of Pittsburgh, Pittsburgh, PA). A431 is a well-characterized vulvar SCC known to over- express EGFR and was used as a positive control. Patient tissues used in the study were obtained from patients undergoing surgery at the University of Pittsburgh Medical Center. Primary HNSCC and normal adjacent mucosa (5 cm away from tumor site) were harvested under the auspices of an Institutional Review Board-approved protocol. Signed informed consent was obtained from each subject. Reagents. For in vitro cell stimulation, recombinant human EGF (Sigma Chemical Co., St. Louis, MO) was used. U73122 (BioMol, Plymouth Meeting, PA) was used to block PLC activity. An inactive analogue of U73122, U73343 (BioMol), was used as a negative control. Specific EGFR tyrosine kinase inhibitor PD153035 was obtained from Calbiochem-Novabiochem Corpora- tion (San Diego, CA). The EGFR blocking antibody C225 was obtained from Imclone Systems Incorporated (New York, NY). Antibodies used included mouse monoclonal anti-PLC -1 (Upstate Biotechnology, Lake Placid, NY), antiphospho-PLC-1 (Cell Signaling Technologies, Beverly, MA), and -actin (Calbiochem-Novabiochem Corporation). Antisense and scrambled PLC-1 oligonucleotides were obtained from MWG Biotech (High Point, NC). The PLC-1 antisense oligonucleotide (5'AGGGGACGCGGCGCCCGCCAT3') is a 21-mer fragment directed against the translation initiation site of human Received 2/12/03; revised 5/20/03; accepted 6/11/03. 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 by NIH Grant 5RO1CA77308 (to J. R. G.), a Veterans Administration Merit Award (to A. W.) and a Lung Specialized Programs of Research Excellence postdoctoral fellowship (to S. M. T.). 2 To whom requests for reprints should be addressed, at Department of Otolaryngol- ogy, The Eye and Ear Institute Building, Suite 500, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 647-5280; Fax: (412) 647-2080; E-mail: jgrandis@pitt.edu. 3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; PLC, phospholipase C; FBS, fetal bovine serum; SCC, squamous cell carcinoma; EGF, epidermal growth factor; IP, inositol phosphate; MMP, matrix metalloproteinase. 5629