Binding with Acquired Resistance to Faslodex ( ICI 182 , 780 ) B , and Cyclic AMP Response Element κ Nuclear Factor-Association of Interferon Regulatory Factor-1 , Nucleophosmin , Updated

To identify genes associated with survival from antiestrogens, both serial analysis of gene expression and gene expression microarrays were used to explore the transcriptomes of antiestrogen-responsive (MCF7/ LCC1) and -resistant variants (MCF7/LCC9) of the MCF-7 human breast cancer cell line. Structure of the gene microarray expression data was visualized at the top level using a novel algorithm that derives the first three principal components, fitted to the antiestrogen-resistant and -responsive gene expression data, from Fisher’s information matrix. The differential regulation of several candidate genes was confirmed. Functional studies of the basal expression and endocrine regulation of transcriptional activation of implicated transcription factors were studied using promoter-reporter assays. The putative tumor suppressor interferon regulatory factor-1 is downregulated in resistant cells, whereas its nucleolar phosphoprotein inhibitor nucleophosmin is up-regulated. Resistant cells also up-regulate the transcriptional activation of cyclic AMP response element (CRE) binding and nuclear factor B (NF B) while down-regulating epidermal growth factor receptor protein expression. Inhibition of NF B activity by ICI 182,780 is lost in resistant cells, but CRE activity is not regulated by ICI 182,780 in either responsive or resistant cells. Parthenolide, a potent and specific inhibitor of NF B, inhibits the anchorage-dependent proliferation of antiestrogen-resistant but not antiestrogen-responsive cells. This observation implies a greater reliance on their increased NF B signaling for proliferation in cells that have survived prolonged exposure to ICI 182,780. These data from serial analysis of gene expression and gene microarray studies implicate changes in a novel signaling pathway, involving interferon regulatory factor-1, nucleophosmin, NF B, and CRE binding in cell survival after antiestrogen exposure. Cells can up-regulate some estrogenresponsive genes while concurrently losing the ability of antiestrogens to regulate their expression. Signaling pathways that are not regulated by estrogens also can be up-regulated. Thus, some breast cancer cells may survive antiestrogen treatment by bypassing specific growth inhibitory signals induced by antagonist-occupied estrogen receptors. INTRODUCTION ERs are nuclear transcription factors, their activities being affected by the nature of the ligand bound and the pattern of genes/proteins expressed within cells (cellular context; Ref. 1). Antiestrogens compete with endogenous estrogens for activation of ER, and induce both cell cycle arrest and apoptosis in responsive cells (2). Neither the genes regulated by antiestrogens that signal to apoptosis nor those genes that confer an acquired antiestrogen resistance have been identified. Nonetheless, antiestrogenic drugs are effective in both premenopausal and postmenopausal breast cancer patients, and in the metastatic and adjuvant settings (3). The most widely used antiestrogen in current clinical practice is the triphenylethylene TAM. Clinical experience with this drug likely now exceeds 10 million patient years. When patients with metastatic disease are selected for treatment based on the ER and PgR content of their tumors, responses are seen in up to 75% of tumors expressing both receptors (2). TAM also reduces the incidence of ER-positive breast cancers in high risk women (4). Other antiestrogens have emerged recently, most notably the benzothiophene Raloxifene and the steroidal ICI 182,780 (Faslodex). Both drugs appear to have significant clinical activity and may have better toxicological profiles when compared with TAM (2). Faslodex has significant activity in TAM-resistant patients (5), consistent with data obtained previously with TAM-resistant human breast cancer cells selected in vitro (6). Despite the utility of antiestrogens, most tumors that initially respond to these drugs will recur and require alternative systemic therapies (2). Unfortunately, the precise mechanisms that confer resistance remain unknown. Change to an antiestrogen-stimulated phenotype has been described in some animal models (6, 7). This phenotype may occur in up to 20% of breast cancer patients but a loss of responsiveness to antiestrogens may be the more common phenotype (2). The expression of mutant ER proteins and splice variants has been reported but the functional role of these in endocrine resistance remains unclear (2). Most tumors acquiring antiestrogen resistance do so while retaining expression of ER (8). Thus, whereas lack of ER expression is a major form of de novo antiestrogen resistance, other mechanisms must be active in most instances of acquired resistance (2). The persistent expression of ER in tumors with acquired resistance suggests that some cells expressing this phenotype may either require ER expression and/or reflect the altered expression of otherwise estrogen-regulated genes. Because ER-mediated transcription is directly affected by antiestrogens, we initially hypothesized that antiestrogen resistance might include perturbations in the patterns of expression and/or regulation of Received 8/31/01; accepted 5/2/02. 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 in part by Public Health Service awards 5R01-CA/AG58022, 5P50CA58185 (to R. C.), 5R33-CA83231 (to Y. W.) from the National Cancer Institute; Department of Defense Awards DAMD17-99-9189 (to K. B. B.), DAMD17-99-1-9191, BC010619, and BC990358 (to R. C.) from the United States Army Medical Research and Materiel Command; and the American Cancer Society IRG-97-1520-01 (to T. C. S.). Technical services also were provided by the Flow Cytometry and Cell Sorting, and Macromolecular Shared Resources funded through Public Health Service Award 2P30CA51008 (Vincent T. Lombardi Cancer Center Support Grant). 2 Present address: Celera Genomics, 45 West Gude Drive, Rockville, MD 20850. 3 Present address: Indiana University, Department of Medicine, Indianapolis, IN 46202. 4 Present address: Laboratory of Clinical Investigation, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224. 5 Present address: United States Patent and Trade Mark Office, Crystal Plaza 3, Washington, DC 20231. 6 To whom requests for reprints should be addressed, at Room W405A Research Building, Vincent T. Lombardi Cancer Center, Georgetown University School of Medicine, 3970 Reservoir Road, NW, Washington, D.C. 20007. Phone: (202) 687-3755; Fax: (202) 687-7505; E-mail: clarker@georgetown.edu. 7 The abbreviations used are: ER, estrogen receptor; CRE, cyclic AMP response element; CCS-IMEM, improved minimal essential medium supplemented with 5% charcoal calf stripped serum; EGF-R, epidermal growth factor receptor; IRF-1, interferon regulatory factor-1; NPM, nucleophosmin; PgR, progesterone receptor; SAGE, serial analysis of gene expression; TAM, Tamoxifen; XBP-1, X-box binding protein-1; FACS, fluorescence-activated cell sorting; NF B, nuclear factor B; EGR-1, early growth response factor-1; TNF , tumor necrosis factor .