HER-2/neu Status Is a Determinant of Mammary Aromatase Activity In vivo : Evidence for a Cyclooxygenase-2-Dependent Mechanism Kotha Subbaramaiah, 1 Louise R. Howe, 2,3 Elisa R. Port, 4 Edi Brogi, 5 Jack Fishman, 1 Catherine H. Liu, 7 Timothy Hla, 7 Clifford Hudis, 6 and Andrew J. Dannenberg 1 Departments of 1 Medicine and 2 Cell and Developmental Biology, Weill Medical College of Cornell University; 3 Strang Cancer Research Laboratory at The Rockefeller University; Departments of 4 Surgery, 5 Pathology, and 6 Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York; and 7 Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut Abstract Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Given the significance of estrogen synthesis in hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 expression. The main objective of this study was to define the interrelationship between HER-2/neu, cyclooxygenase-2 (COX-2), and aroma- tase in mammary tissue. Mammary aromatase activity and prostaglandin E 2 (PGE 2 ) levels were increased in mice with mammary-targeted expression of a COX-2 transgene. In vitro , overexpressing COX-2 caused both increased PGE 2 production and aromatase activity, effects that were suppressed by celecoxib, a selective COX-2 inhibitor. Previously, we found that overexpression of HER-2/neu was associated with increased levels of COX-2 in human breast cancers. Here, we show that overexpression of HER-2/neu is also associated with increased aromatase activity. These results suggested the possibility that COX-2 was the functional intermediate linking HER-2/neu and aromatase. Consistent with this idea, COX-2 deficiency led to a gene dose-dependent reduction in mam- mary aromatase activity in a HER-2/neu transgenic mouse model. Complementary in vitro studies showed that HER-2/ neu–mediated induction of PGE 2 synthesis and aromatase activity were suppressed by inhibiting COX-2. Collectively, our data indicate that COX-2 is the functional intermediate linking HER-2/neu and aromatase and suggest that inhibitors of PGE 2 synthesis will suppress estrogen biosynthe- sis in breast tissue. (Cancer Res 2006; 66(10): 5504-11) Introduction Approximately 60% of breast cancer patients have hormone- dependent breast cancers, which express estrogen receptors and require estrogen for tumor growth. Estrogens are synthesized from androgens in a reaction catalyzed by the cytochrome P450 aromatase, encoded by the CYP19 gene (1). In postmenopausal women, peripheral aromatization in adipose tissues is largely responsible for estrogen production, and in particular mammary adipose tissue is considered an important local estrogen source. Aromatase activity is elevated in tumor stroma (2), suggesting that up-regulation of aromatase occurs during carcinogenesis and contributes to tumor growth (3). Thus, estrogen deprivation is a commonly used approach for the prevention and treatment of hormone-dependent breast cancer (4, 5). Given the significance of estrogen synthesis in hormone- dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 expression. The aromatase CYP19 gene is normally expressed from promoter I.4 in breast adipose tissue, but in breast cancers and cancer-proximal adipose tissue expression occurs predominantly from promoters I.3 and II (6–8). Transcription from promoter II is stimulated by cyclic AMP (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB)–dependent signaling (9, 10). Several findings suggest that cyclooxygenase (COX)–derived prostaglandin E 2 (PGE 2 ) may act locally in breast tissue to stimulate CYP19 expression and aromatase activity. PGE 2 is a potent inducer of CYP19 transcription and aromatase activity via a cAMP-dependent mechanism in breast adipose stromal cells in vitro (9, 10). Moreover, positive correlations have been detected between COX and aromatase expression in human breast cancer specimens (11, 12). Finally, aspirin, an inhibitor of COX-mediated PGE 2 synthesis, was recently found to protect against hormone receptor– positive but not hormone receptor–negative breast cancers (13). Together, these data suggestthat COX-derived PGE 2 may play a role in hormone-dependent breast carcinogenesis. There are two isoforms of COX, designated COX-1 and COX-2, which catalyze the first step in the synthesis of PGE 2 from arachidonic acid. COX-1 is constitutively expressed in most tissues and seems to fulfill housekeeping functions (14, 15). In contrast, COX-2 is not detected in most normal tissues. However, it is induced by a variety of mitogenic and inflammatory stimuli (16–22), resulting in enhanced amounts of prostaglandins in neoplastic and inflamed tissues (23–25). COX-2 is overexpressed in transformed mammary epithelial cells (20), the preinvasive lesion ductal carcinoma in situ (DCIS; refs. 26–28) and in a significant proportion of breast cancers, particularly in those that overexpress HER-2/neu (26, 29–31). The results of numerous genetic and pharmacologic preclinical studies suggest that COX-2 is a bona fide molecular target for inhibiting breast carcinogenesis, including HER-2/neu –overexpressing cancers (32–36). Collectively, the above findings suggest that nonsteroidal anti- inflammatory drugs, inhibitors of COX activity, should inhibit PGE 2 -mediated induction of estrogen biosynthesis in breast tissue. However, no direct causal relationship has been shown between COX expression and aromatase activity in vivo . Therefore, the aim of this study was to analyze the functional relationship between COX-2 and aromatase in vivo . Additionally, because COX-2 expression correlates with HER-2/neu overexpression in human Requests for reprints: Kotha Subbaramaiah, New York Presbyterian Hospital- Cornell, 525 East 68th Street, Room F-203A, New York, NY 10021. Phone: 212-746-4402; Fax: 212-746-4885; E-mail: ksubba@med.cornell.edu or Andrew J. Dannenberg, New York Presbyterian Hospital-Cornell, 525 East 68th Street, Room F-206, New York, NY 10021. Phone: 212-746-4403; Fax: 212-746-4885; E-mail: ajdannen@med.cornell.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-4076 Cancer Res 2006; 66: (10). May 15, 2006 5504 www.aacrjournals.org Research Article Research. on November 27, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from