Cyclooxygenase-2 Inhibition Suppresses A v B 6 Integrin–Dependent Oral Squamous Carcinoma Invasion Maria L. Nystrom, 1 Diana McCulloch, 1 Paul H. Weinreb, 3 Shelia M. Violette, 3 Paul M. Speight, 4 John F. Marshall, 1 Ian R. Hart, 1 and Gareth J. Thomas 1,2 1 Tumour Biology Laboratory, Cancer Research UK Clinical Centre, Queen Mary’s University, Charterhouse Square; 2 Department of Histopathology, University College London, London, United Kingdom; 3 Biogen Idec, Cambridge, Massachusetts; and 4 Department of Oral Pathology, School of Dentistry, University of Sheffield, Sheffield, United Kingdom Abstract Worldwide oral squamous cell carcinoma (OSCC) represents about 5.5% of all malignancies, with f30,000 new cases each year in the United States. The integrin A v B 6 and the enzyme cyclooxygenase-2 (COX-2) are implicated in OSCC progression and have been suggested as possible therapeutic targets. Each protein also is reported to identify dysplasias at high risk of malignant transformation, and current clinical trials are testing the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) at preventing OSCC development. Given the probable increased expression of A v B 6 and COX-2 in OSCC and the inhibition of several integrins by NSAIDs, we investigated whether NSAIDs affected A v B 6 -dependent cell functions. We found that expression of both A v B 6 and COX-2 was signifi- cantly higher in OSCC compared with oral epithelial dyspla- sias. Neither protein preferentially identified those dysplastic lesions that became malignant. Using OSCC cell lines, modified to express varying levels of A v B 6 , we assessed the effect of COX-2 inhibition on cell invasion. We found that the COX-2 inhibitor NS398 inhibited specifically A v B 6 -dependent, but not A v B 6 -independent, OSCC invasion in vitro and in vivo , and this effect was modulated through prostaglandin E 2 (PGE 2 )–dependent activation of Rac-1. Transient expression of constitutively active Rac-1, or addition of the COX-2 metabolite PGE 2 , prevented the anti-invasive effect of NS398. Conversely, RNA interference down-regulation of Rac-1 inhibited A v B 6 -dependent invasion. These findings suggest that COX-2 and A v B 6 interact in promoting OSCC invasion. This is a novel mechanism that, given the ubiquity of A v B 6 expression by head and neck cancers, raises the possibility that NSAIDs could protect against OSCC invasion. (Cancer Res 2006; 66(22): 10833-42) Introduction Cyclooxygenases (COX) catalyze the key step in prostanoid and thromboxane biosynthesis and are targets of nonsteroidal anti- inflammatory drugs (NSAID). Two human isoforms exist: COX-1, expressed constitutively in most mammalian cells, generates prostaglandins necessary for normal physiologic function, whereas COX-2, normally undetectable, is induced rapidly by stimuli, including cytokines, oncogenes, and tumor promoters (1, 2). Elevated COX-2 expression occurs in many carcinomas, including oral squamous carcinoma (OSCC), where it contributes to tumor progression (1–3). Transgenic female mice overexpressing COX-2, for example, have a high frequency of breast dysplasia and metastatic tumors (4), whereas genetic inactivation of COX-2 in a murine model of familial adenomatous polyposis reduced both the size and number of intestinal polyps (5). The epithelial integrin a v h 6 is a receptor for fibronectin, vitronectin, tenascin, and the latency-associated peptide (LAP) of transforming growth factor-h (e.g., ref. 6). Generally found to have minimal expression in adult epithelium, a v h 6 is up-regulated during epithelial remodeling (e.g., wound healing) and tumorigen- esis. Aberrant a v h 6 expression occurs in numerous carcinoma types, particularly OSCC (7–11). Moreover, increased expression of both a v h 6 and COX-2 have been reported in oral dysplastic epithelium, suggesting a role in tumor progression (12, 13). NSAIDs have marked antitumor activity via COX-2 inhibition. Selective COX-2 inhibitors are chemopreventive in animal models of colon, bladder, and breast cancer (1, 2), whereas epidemiologic studies indicate that NSAIDs reduce breast, esophageal, and colon cancer mortality (1, 2). The effects of NSAIDs on tumor growth and progression are likely to be through multiple mechanisms, in- cluding indirect effects on angiogenesis and inflammation, as well as direct effects on tumor cell proliferation and apoptosis (1, 2). Particularly interesting are studies where NSAIDs inhibit tumor cell adhesion, migration, and invasion (1, 2, 14). These changes may be mediated via effects on various integrins (15, 16). For example, aspirin and indomethacin inhibit activation of the platelet integrin a IIb h 3 (17) whereas Dormond et al. inhibited endothelial cell migration and angiogenesis using the selective COX-2 inhibitor NS398, effects that were modulated through inhibition of the a v h 3 - dependent activation of Rac-1 and Cdc42 (18). This may be a reciprocal effect because integrin-mediated adhesion can contrib- ute to stabilization of COX-2 protein levels (19). Given that both a v h 6 and COX-2 are expressed in OSCC and have been reported to promote invasion (20–22), and that NSAIDs may inhibit integrin function, we determined whether NSAIDs could inhibit a v h 6 -mediated invasive activity. Materials and Methods Immunohistochemistry. Antibodies used were anti-cytokeratin, AE1/ AE3 (1:50; DAKO, High Wycombe, United Kingdom); anti-a v h 6 , 6.2G2 (0.5 Ag/mL; Biogen Idec, Cambridge, MA); or anti-COX-2 (1:500; Cayman Chemical Co., Ann Arbor, MI). Antigen retrieval varied according to primary antibody: 0.1% a-chymotrypsin, 0.1% calcium chloride (pH 7.8) for Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M.L. Nystrom and D. McCulloch contributed equally to this work. M.L. Nystrom is a Medical Research Council Clinical Training Fellow. G.J. Thomas is a Health Foundation Senior Clinical Fellow. Requests for reprints: Ian R. Hart or Gareth J. Thomas, Tumour Biology Laboratory, Cancer Research UK Clinical Centre, Queen Mary’s University, Charterhouse Square, London EC1M 6BQ, United Kingdom. Phone: 44-20-70140409/ 0400; Fax: 44-20-70140401; E-mail: ian.hart@cancer.org.uk or gareth.thomas@cancer. org.uk. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1640 www.aacrjournals.org 10833 Cancer Res 2006; 66: (22). November 15, 2006 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/66/22/10833/2555464/10833.pdf by guest on 21 December 2023