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Molecular Cancer Therapeutics 151 Distinct gene expression patterns in a tamoxifen-sensitive human mammary carcinoma xenograft and its tamoxifen-resistant subline MaCa 3366/TAM Michael Becker,1 Anette Sommer,2 of acquired tamoxifen resistance. Finally, genes whose Jo¨rn R. Kra¨tzschmar,2 Henrik Seidel,2 expression profiles are distinctly changed between the Hans-Dieter Pohlenz,2 and Iduna Fichtner1 two xenograft lines will be further evaluated as potential targets for diagnostic or therapeutic approaches of 1 Max-Delbrueck-Center for Molecular Medicine, Experimental tamoxifen-resistant breast cancer. [Mol Cancer Ther Pharmacology, and 2Research Laboratories, Schering AG, Berlin, Germany 2005;4(1):151–68] Abstract Introduction The reasons why human mammary tumors become Breast cancer is the most common type of cancer in women resistant to tamoxifen therapy are mainly unknown. of the Western world. Due to advances in early detection Changes in gene expression may occur as cells acquire and treatment, breast cancer survival rates have increased resistance to antiestrogens. We therefore undertook a com- markedly over the past decades. After surgery, estrogen parative gene expression analysis of tamoxifen-sensitive receptor a (ERa)–positive breast cancer is usually treated and tamoxifen-resistant human breast cancer in vivo with endocrine therapy. Tamoxifen, a nonsteroidal anti- models using Affymetrix oligonucleotide arrays to analyze estrogen, also termed selective estrogen receptor modula- differential gene expression. Total RNAs from the tamox- tor, is the first-line therapy for premenopausal and, until ifen-sensitive patient-derived mammary carcinoma xeno- recently, also for postmenopausal hormone receptor– graft MaCa 3366 and the tamoxifen-resistant model MaCa positive women (1). For postmenopausal women, three 3366/TAM were hybridized to Affymetrix HuGeneFL and different aromatase inhibitors are now available that might to Hu95Av2 arrays. Pairwise comparisons and clustering replace tamoxifen as first-line therapy in the future. algorithms were applied to identify differentially expressed Tamoxifen is effective both as adjuvant therapy and for genes and patterns of gene expression. As revealed by advanced disease of hormone-responsive breast cancer and cluster analysis, the tamoxifen-sensitive and the tamoxi- can prevent breast cancer in high-risk patients (2). In many fen-resistant breast carcinomas differed regarding their cases, however, therapies fail and women die from gene expression pattern. More than 100 transcripts are recurrent, endocrine-resistant breast cancer. Prognosis of changed in abundance in MaCa 3366/TAM as compared hormone-dependent breast cancer as well as treatment with MaCa 3366. Among the genes that are differentially strategies are mainly determined by the presence of the expressed in the tamoxifen-resistant tumors, there are ERa and the progesterone receptor (PR). Two thirds of the several IFN-inducible and estrogen-responsive genes, and patients who present with breast cancer are ERa positive genes known to be involved in breast carcinogenesis. The (3). Treatment of estrogen-dependent breast cancer with an genes neuronatin (NNAT) and bone marrow stem cell antiestrogen like tamoxifen inhibits tumor growth. ERa- antigen 2 (BST2) were sharply up-regulated in MaCa and PR-positive breast cancer have a better response rate 3366/TAM. The differential expression of four genes than ERa- and PR-negative breast cancers (3). However, a (NNAT, BST2, IGFBP5, and BCAS1) was confirmed by large number of originally tamoxifen-sensitive tumors Taqman PCR. Our results provide the starting point for develop resistance after several months of treatment while deriving markers for tamoxifen resistance by differential still expressing the ERa (4). gene expression profiling in a human breast cancer model Antagonism of tamoxifen has been attributed to the antiestrogenic activity of tamoxifen in which the active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OHT), competes with E2 for binding to the ERa. Activation of the transcriptional activation domain AF-2 of the ERa, but Received 8/18/04; revised 11/2/04; accepted 11/8/04. not AF-1, is prevented by 4-OHT because coactivators can a The costs of publication of this article were defrayed in part by the no longer bind to the antagonist-occupied ER (5). Clinical payment of page charges. This article must therefore be hereby marked manifestation of tamoxifen resistance is now often inter- advertisement in accordance with 18 U.S.C. Section 1734 solely to preted as a manifestation of increased tamoxifen agonism indicate this fact. and as a switch from tamoxifen-dependent growth inhibi- Note: M. Becker and A. Sommer contributed equally to this work. tion to growth stimulation (6, 7). The phenomenon of Requests for reprints: Michael Becker, Max-Delbrueck-Center for Molecular Medicine, Robert-Ro¨ssle-Strasse 10, D-13092 Berlin, Germany. Phone: 49- tamoxifen resistance is poorly understood and genetic 30-9406-2702; Fax: 49-30-9406-3823. E-mail: [email protected] mechanisms have been proposed, but mutations in the ERa Copyright C 2005 American Association for Cancer Research. are rare events in both patients with tamoxifen-resistant Mol Cancer Ther 2005;4(1). January 2005 Downloaded from mct.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. 152 Gene Expression Analysis of Tamoxifen-Resistant Xenograft breast cancer and in various cell culture models of genes of tamoxifen resistance in a diagnostic or prognostic tamoxifen resistance (8, 9). In the MaCa 3366/TAM approach. By using clustering algorithms as well as xenograft model, mutations in the ERa ligand binding pairwise comparisons of sample groups, we focussed on domain were not identified (10, 11). Tamoxifen resistance those genes, which distinguish the tamoxifen-sensitive most likely is a multicausal phenomenon. A disturbance of from the tamoxifen-resistant phenotype. growth and survival pathways namely of growth factors, their receptors, extracellular proteins, proteases like kalli- krein 10, immediate-early genes, transcription factors, cell Materials and Methods cycle regulators, signal transduction molecules like Animal Experiment BCAR1/p130Cas, phosphorylation of the ERa by protein Animals. For the animal experiments, 50 female nude kinase A, and alterations in the uptake, retention, and mice (Bom: NMRI-nu/nu) per xenograft experiment, ages 4 metabolism of tamoxifen might all contribute to tamoxifen to 6 weeks and weighing 20 to 24 g, were used. Breeding resistance (12–18). Clues to the mechanisms of tamoxifen and keeping conditions have been described (38). All resistance could therefore be gained from an understanding animal experiments were done according to the United of the numerous effects that tamoxifen produces at the gene Kingdom Coordinating Committee on Cancer Research expression level. Guidelines for the Welfare of Animals in Experimental We decided to approach the question of tamoxifen Neoplasia and with the permission of the responsible local resistance in a xenograft system. Xenografts of human authorities (G V247/98). tumors resemble the clinical situation much more closely Tumor Transplantation. The s.c. transplantation of the than cell lines do (11). The xenograft tumor line MaCa tumor fragments (size, 4  4  4mm3) was done under 3366/TAM is one of the very few in vivo preclinical models Radenarkon anesthesia (40 mg/kg i.p. Etomidat, Asta in which antiestrogen resistance was induced in a clinically Medica, Frankfurt, Germany). The diameter of the tumors adapted manner. By direct transplantation of a ductal was measured once weekly using a caliper-like mechanical invasive carcinoma with moderate differentiation from a instrument and the tumor volume (V)wascalculated postmenopausal woman onto nude mice, the xenograft according to the empirical equation V = (length  width2)/2. tumor line MaCa 3366 was established (19). To study The median volumes of each group were normalized to the tamoxifen resistance in an in vivo model, the tamoxifen- initial tumor volume resulting in the relative tumor resistant xenograft tumor line MaCa 3366/TAM was volume. In all the experiments, tumor-bearing mice developed by treatment of the tamoxifen-sensitive parental received estradiol supplementation [estradiol valeriate human xenograft tumor MaCa 3366 with the antiestrogen (E2D), 0.5 mg/kg once/wk i.m.]. This supplementation tamoxifen during successive passaging over 2 years (10, 11). leads to physiologic levels of serum E2 (25-984 pg/mL) that MaCa 3366 and MaCa 3366/TAM are both ERa and PR are comparable to the human situation (25–600 pg/mL positive. In both xenograft tumor lines, the PR is inducible depending on the follicular phase). by E2 indicating that the ERa-dependent transcriptional Substances. The following substances were used: E2D regulation is still intact. (Jenapharm, Jena, Germany) and tamoxifen (Sigma, In recent years, several gene expression profiling studies Chemie GmbH, Taufkirchen, Germany). were performed to identify genes that are differentially Treatment Modalities. Two independent experiments expressed in human breast cancer which then allow to were done: All MaCa 3366 and MaCa 3366/TAM trans- classify tumors and to predict outcome (20–27). In in vitro planted animals received E2D (0.5 mg/kg) injections once a breast cancer cell culture models, the influence of estrogen week. The last passage of MaCa 3366/TAM before the start or antiestrogen treatment

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