Distinct Gene Expression Patterns in a Tamoxifen-Sensitive Human Mammary Carcinoma Xenograft and Its Tamoxifen-Resistant Subline Maca 3366/TAM

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

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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 kallikrein 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 Â 4 Â 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 Â 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 was also analyzed (28–37). of an experiment was cultivated without tamoxifen It was therefore our aim to compare the gene expression treatment. MaCa 3366 and MaCa 3366/TAM were either profile of the parental tamoxifen-sensitive mammary left untreated, or treated with 50 mg/kg tamoxifen 24 hours carcinoma xenograft model MaCa 3366 and its tamoxifen- before sacrifice (short-term tamoxifen treatment). An resistant derivative MaCa 3366/TAM on a genome-wide additional group of MaCa 3366/TAM xenograft animals scale to analyze the molecular events that lead to the was treated once a week with 50 mg/kg tamoxifen during tamoxifen-resistant phenotype in an in vivo system. By the course of the experiment starting on day 1 (permanent comparing the gene expression pattern of MaCa 3366, tamoxifen treatment; Fig. 1). As permanent tamoxifen untreated or after short-term tamoxifen treatment, and treatment of MaCa 3366 led to complete remission of MaCa 3366/TAM, either untreated, after short-term or tumors, this group could not be included in the analysis. permanent tamoxifen treatment, we expected to identify From histologic and fluorescence-activated cell sorting genes which respond to short-term tamoxifen treatment analyses of MaCa 3366 and MaCa 3366/TAM, the amount and those which distinguish the tamoxifen-sensitive from of murine tissue in a human breast cancer xenograft tumor the tamoxifen-resistant phenotype. Different types of gene was estimated to be as low as 5% to 10%.3 expression profiles can be envisioned which might either indicate an involvement of a particular gene in the biological process and the mechanism of tamoxifen resistance or which can potentially be exploited as marker 3 I. Fichtner and M. Becker, unpublished observation.

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Palo Alto, CA). The cRNA was prepared as described above (39). The HuGeneFL and Hu95Av2 arrays (Affyme- trix, Santa Clara, CA) were hybridized, washed, and stained with streptavidin-phycoerythrin (Moleculer Probes, Eugene, OR), biotinylated anti-streptavidin (Vector Labo- ratories, Burlingame, CA) and streptavidin-phycoerythrin according to the standard antibody amplification protocol for eukaryotic targets. The arrays were scanned with a confocal scanner from Affymetrix at 570 nm. Data Analysis The Affymetrix arrays were normalized by employing the Maximum Order Preserving Set method. This method identifies a subset of genes which does not change its expression between two experiments. The intensity scatter plot for all genes from this restricted set is approximated by a nonlinear regression function, which is then used for normalization as described above (39). The normalized expression data sets were loaded into the CoBi database (Genedata, Basel, Switzerland) and analyzed with the Genedata Expressionist software version 5.0 (40, 41). The expression data were filtered with the coefficient of variation and on the variant data set three different fold change–based two-group tests (intergroup gap, valid value proportions, and ratio of means) were done. A ratio of means is the ratio of the mean expression values for two sample groups under investigation. A group mean is calculated as the exponentialized arithmetic mean of all logarithmized expression values within a group. This is identical to the geometric mean of all expression values. For the valid value proportions analysis, all possible pairwise combinations of a sample from the first sample group and another sample from the second sample group are created. If the first group contained m samples and the second Figure 1. Experimental workflow. Five nude mice each were trans- group contained n samples, then there were m Â n such planted in the flank with the human breast cancer xenograft model MaCa sample pairs. Subsequently, for each sample pair, the ratio 3366 and MaCa 3366/TAM and all animals were supplemented with E2D (0.5 mg/kg) injections once a week. MaCa 3366 and MaCa 3366/TAM of their corresponding expression values is calculated and were either left untreated, or treated with 50 mg/kg tamoxifen (TAM)24h compared with a threshold which is an analysis variable. before sacrifice (short-term TAM treatment), and MaCa 3366/TAM were The percentage of sample pairs which pass this threshold is treated once per week with 50 mg/kg TAM in the course of the experiment determined. Finally, this type of analysis returns all probe (permanent TAM treatment). Two independent xenograft experiments were conducted. RNA was isolated from the tumors and RNA from the five sets for which the proportion of sample pairs with above- animals per treatment group was pooled. The cRNA from the first threshold expression ratios exceeds a given percentage xenograft experiment was hybridized to Affymetrix HuGeneFL arrays, which is another analysis variable. The intergroup gap the cRNA from the second experiment to Hu95Av2 arrays. Both data sets were subjected to supervised and unsupervised analyses that were done method identifies probe sets which have consistently with the Expressionist software (Genedata). higher expression values in one of the two sample groups. If expression values in the first sample group are smaller than in the second sample group, then the intergroup gap is Extraction of RNA the ratio between the smallest value of the second group Tumor tissue samples (size, 2 Â 2 Â 2mm3) were taken and the largest value of the first group. The intergroup gap from the sacrificed animals. Samples were snap frozen and method then returns all probe sets for which this ratio is stored in liquid nitrogen until use. Total RNA of tumor above a given threshold. Probe sets with overlapping samples was prepared using the RNeasy Mini Kit (Qiagen, ranges of expression values are never returned by this type Hilden, Germany) according to the manufacturer’s recom- of analysis. Statistical tests could not be applied because mendations. A DNase I (Qiagen) digestion step was only two or three samples were present in each sample included to eliminate DNA contamination. group. Affymetrix DNA Chip Hybridization Probe Sets. Profiling human genes expressed in a human Isolated total RNA was checked for integrity and concen- breast cancer–derived xenograft model transplanted onto tration using the RNA 6000 Assay and RNA LabChips on nude mice using the human-specific Affymetrix arrays the Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., HuGeneFL and Hu95Av2, will mostly uncover genes

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specifically expressed in human cancer cells in xenograft (1.3-fold), valid value proportions 40% (40%), ratio of tumors that might comprise a mixture of human- and means 1.8-fold (1.6-fold)] were used to identify differen- mouse-derived cells. Using the human-specific Affymetrix tially expressed probe sets between MaCa 3366/TAM arrays in contrast to cDNA arrays has the advantage that (untreated, short-term, and permanent tamoxifen treat- 20 independent probes as perfect match oligonucleotides ment) and MaCa 3366 (untreated, short-term tamoxifen interrogate a particular gene. Unless a murine gene is 100% treatment). [Values in brackets for HuGeneFL array.] Those homologous to a human gene, this provides very high 154 [50] probe sets overlapping (intersection) and those 362 specificity compared with a continuous stretch of DNA on [140] probe sets identified with any of the three indepen- a cDNA array. The probe sets are usually situated either at dently conducted, fold change–based tests were further the extreme 3V end of the cDNA or in the 3V untranslated subjected to a k-means clustering resulting in two groups region of the human genes. In the latter case, there is a very consisting of 57 [37] and 144 [125] probe sets up-regulated low probability that a human-specific probe set detects a and 97 [13] and 218 [67] down-regulated, respectively, in all murine gene because the homology between human and MaCa 3366/TAM compared with all MaCa 3366 tumors. To murine sequence is usually very low in the 3V untranslated compare differentially expressed genes on the two array region. In addition, the presence of a set of 20 mismatch formats, the 144 and 125 up-regulated and 218 and 67 oligonucleotides provides a further degree of specificity as down-regulated probe sets from the Hu95Av2 and HuGe- only those probe sets are incorporated in the determina- neFL array, respectively, were subjected to manual inspec- tion of a condensed signal intensity for which the signal tion combined with a BLAST analysis of the genes intensity on the perfect match is larger than on the mis- corresponding to the probe set identifiers. Differential match oligonucleotide. The probe sets on the two array expression of several genes was detected with more than generations HuGeneFL and Hu95Av2 were changed by one probe set. Nonredundant genes that are regulated and Affymetrix such that different probe sets are present on present on both array types and genes present on only one both arrays. If genes are interrogated on both types of array, but strongly regulated, are displayed. arrays by independent probe sets or if more than one Quantitative Real-time PCR probe set interrogates a particular gene on one array then Six genes (BST2, NNAT, IGFBP5, ESRRG, PTK6, and the differential expression of human genes is further BCAS1) were selected and analyzed by quantitative real- corroborated. time reverse transcription-PCR based on TaqMan-chemis- Unsupervised Analysis. For the unsupervised analysis, try (Table 1). Hydroxymethylbilane synthase (HMBS) was genes with invariant expression were filtered out by the used as housekeeping gene (Table 1). Predeveloped coefficient of variation analysis. HuGeneFL data were reagents containing a specific primer pair/probe mix, filtered with a coefficient of variation (CV) z 0.03175 premixed TaqMan Reverse Transcription Reagents and resulting in a selection of 503 genes. For the analysis of TaqMan Universal MasterMix were obtained from Ap- Hu95Av2 data, a CV z 0.04050 selected 538 genes. The CV plied Biosystems GmbH (Weiterstadt, Germany). For was chosen so that a manageable number of probe sets was BCAS1 and HMBS, new primer pair/probe combinations obtained. k-Means clustering was applied and genes were were designed by the Assay-by-Design-service at Applied sorted into 10 different clusters. Biosystem using the template sequences (Table 1). Pooled Supervised Analysis. To select for variant genes a CV z RNA from n = 5 animals from the five treatment groups 0.001091 (CV z 0.001097 for HuGeneFL array) was applied obtained from the two independent xenograft experi- to the data derived from the Hu95Av2 array over all ments was reverse transcribed with the Reverse Tran- hybridization samples and 4,887 (2,305 for HuGeneFL scription Reagents (Applied Biosystems) and contained array) probe sets were selected from a total of 12386 probe 200 ng RNA in a final volume of 60 AL. The RT reaction sets (6,574 for HuGeneFL array). Next, three different fold was carried out at 25jC for 10 minutes, 48jC for 30 change–based two-group tests [intergroup gap >1.5-fold minutes, and 95jC for 5 minutes. The PCR was done in

Table 1. TaqMan assays

Gene symbol Gene description Accession ID Assay ID

BST2 Bone marrow stromal cell antigen 2 NM_004335 Hs00171632_m1 NNAT Neuronatin NM_005386 Hs00193590_m1 IGFBP5 Insulin-like growth factor binding protein 5 NM_000590 Hs00181213_m1 ESRRG Estrogen-related receptor g NM_001438 Hs00155006_m1 PTK6 Protein tyrosine kinase 6 NM_005975 Hs00178742_m1 BCAS1 Breast carcinoma amplified sequence 1 NM_003657 Assay-by-Design HMBS Hydroxymethylbilane synthase NM_000190 Assay-by-Design

NOTE: The gene symbol is the most commonly used abbreviation, usually from OMIM. The accession ID is the National Center for Biotechnology Information RefSeq or reference sequence for this gene. The assay ID is the unique name of the predeveloped or TaqMan assay from Applied Biosystems.

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96-well microtiter plates in an ABI Prism 7700 Sequence MaCa 3366/TAM either untreated, after short-term or Detection System (Applied Biosystems). The reaction permanent tamoxifen treatment, we expect to identify mixture consisted of 10 AL Taqman Universal MasterMix, genes that respond to short-term tamoxifen treatment and 1 AL primer/probe-mix, and 2 AL of the RT reaction mix genes that distinguish the tamoxifen-sensitive from the in a final volume of 20 AL. Following an initial step at tamoxifen-resistant phenotype. 95jC for 10 minutes for denaturation of the DNA and We adopted two different approaches in an attempt to activation of the Taq enzyme, 45 cycles consisting of define gene expression patterns segregating with a denaturation at 95jC for 15 seconds and annealing/ tamoxifen-resistant compared with a tamoxifen-sensitive extension at 60jC for 1 minute were done. Each sample xenograft phenotype (Fig. 1). For unsupervised analysis, k- was tested in triplicate. means clustering with positive correlation as distance The relative expression of each gene was determined on measure was applied to the variant gene set of 503 genes the basis of the threshold cycle (CT value). With cDNA on the HuGeneFL array and 538 genes on the Hu95Av2 from testis, placenta, and prostate (Clontech, Palo Alto, array and genes were sorted into 10 different clusters. CA) standard curves were generated. For each CT value, Profiles for the 10 clusters on the HuGeneFL and on the a corresponding ng amount of cDNA was calculated. With Hu95Av2 array are depicted in Fig. 3A and B, respectively. the housekeeping gene HMBS the quantity of cDNA was When the two independent xenograft experiments were normalized. Then, the normalized target gene expression hybridized to two different array types, the corresponding was divided by the normalized gene expression value of clusters on the two array types were identified first and the calibrator (i.e. untreated MaCa 3366 xenograft) to afterwards genes differentially expressed in these clusters obtain the fold change of the gene in the treatment groups were analyzed. Cluster 5 on HugeneFL corresponds to relative to the untreated MaCa 3366 xenograft. The SD of cluster 9 on Hu95Av2 and contains genes, which are solely the normalized target gene expression relative to HMBS is sharply up-regulated in MaCa 3366/TAM treated perma- calculated from the initial SDs of the target gene and nently with tamoxifen. Similarly, cluster 8 on HuGeneFL HMBS. corresponds to cluster 3 on Hu95Av2 and contains genes that are up-regulated in all three MaCa 3366/TAM xenografts independent of tamoxifen or length of tamoxifen Results treatment. Moreover, the profile of cluster 4 in the Originating from the tamoxifen-sensitive human mammary HuGeneFL data set corresponds to the profile of cluster carcinoma xenograft MaCa 3366, the tamoxifen-resistant 5 in the Hu95Av2 data set and contains genes that are model MaCa 3366/TAM was successfully established by the treatment of tumor-bearing nude mice with tamoxifen for 2 years during routine passaging. Tumor fragments were transplanted s.c. to female nude mice, which were supplemented with E2D to support tumor growth. For the analysis of differential gene expression, the five treatment groups were MaCa 3366 and MaCa 3366/TAM untreated and with short-term tamoxifen treatment, and MaCa 3366/ TAM under permanent tamoxifen treatment (Fig. 1). In order to distinguish the effects of short-term tamoxifen treatment on gene expression from the effects of permanent tamoxifen treatment on resistance, the MaCa 3366 and the MaCa 3366/TAM xenograft mice were treated once with tamoxifen 24 hours before sacrifice (short-term tamoxifen treatment). The tumor growth curve of the five treatment groups from the second xenograft experiment are displayed (Fig. 2). Although MaCa 3366/TAM tumors grow more slowly than MaCa 3366 tumors growth of MaCa 3366/TAM tumors is independent of tamoxifen treatment. MaCa 3366 tumors treated with tamoxifen showed complete remission (data not shown). Tumor samples from both xenograft lines were used for differential gene expression analysis. Total RNA was Figure 2. Tumor growth curves of MaCa 3366 and MaCa 3366/TAM xenografts on nude mice (n = 5) supplemented with estradiol valeriate isolated from the tumor tissues, RNA from five animals (E2D, 0.5mg/kg/wk i.m.). MaCa 3366 and MaCa 3366/TAM were either per group was pooled, and the cRNA from the first and left untreated, or treated with 50 mg/kg TAM 24 h before sacrifice (short- second xenograft experiments was hybridized to Affyme- term TAM treatment), and MaCa 3366/TAM were treated once per week trix HuGeneFL and Hu95Av2 arrays, respectively (Fig. 1). with 50 mg/kg TAM treatment in the course of the experiment (permanent TAM treatment). Treatment was initiated 1 wk after tumor transplantation By comparing the gene expression pattern of MaCa 3366 and continued until the end of the experiment. Relative tumor volume untreated or after short-term tamoxifen treatment and (rtv) F SD over time is shown.

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Figure 3. On the variant 503 genes from the HuGeneFL array and the 538 variant genes on the Hu95Av2 array, k-means clustering was applied to obtain 10 clusters. Screenshots from the Expressionist software (Genedata). MaCa 3366 (A) untreated, (B) short-term TAM treated; MaCa 3366/TAM (C) untreated, (D) short-term TAM treated, and (E) permanently TAM treated.

up-regulated with increasing exposure of the MaCa3366 sure to tamoxifen (cluster 5: 51 genes). Surprisingly, we also xenografts to tamoxifen. However, as probe sets and found genes that are only up-regulated after permanent expression values vary between the data sets on the tamoxifen treatment in MaCa 3366/TAM (cluster 9: 33 HuGeneFL and Hu95Av2 array, a complete concordance genes). of clusters cannot be expected. Applying k-means clustering To identify differentially expressed probe sets between on the Hu95Av2 array variant data set consisting of 538 MaCa 3366/TAM (untreated, short-term, and permanent genes, the following prominent gene clusters were identi- tamoxifen treatment) and MaCa 3366 (untreated and short- fied: (1) genes that are remarkably up-regulated in all MaCa term tamoxifen treated), a supervised analysis was done 3366/TAM xenografts (cluster 3: 88 genes), (2) genes that and three different fold change-based tests were applied are noticeably down-regulated in all MaCa 3366/TAM (see Material and Methods) resulting in four groups with 57 xenografts (cluster 1: 104 genes), (3) genes that are up- (37) probe sets up-regulated and 97 (13) down-regulated on regulated by short-term tamoxifen treatment in MaCa 3366 stringent criteria and 144 (125) probe sets up-regulated and and MaCa 3366/TAM (cluster 6: 114 genes, cluster 7: 29 218 (67) down-regulated on less stringent criteria, respec- genes, cluster 10: 44 genes), (4) genes that are down- tively, in all tamoxifen-resistant compared with all tamox- regulated by short-term tamoxifen treatment in MaCa 3366 ifen-sensitive samples on the Hu95Av2 array (values for and MaCa 3366/TAM (cluster 2: 16 genes), and (5) genes HuGeneFL in brackets). The redundancy (i.e. more than that are increasingly up-regulated in the MaCa 3366 and one probe set mapping to one particular gene) was MaCa 3366/TAM xenograft tumors with extended expo- removed by a BLAST analysis of the corresponding gene

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identifiers as supplied by Affymetrix, and the Online reproducibility of the two independent xenograft experi- Mendelian Inheritance in Man (OMIM) description was ments and the reproducibility of results even if different attributed to each probe set. To compare differentially array generations (i.e., HuGeneFL and Hu95Av2 with expressed genes on the two different array formats, the 144 different probe sets are used). up-regulated probe sets from the Hu95Av2 and 125 up- IFN-Inducible Genes. Very prominently, among the regulated probe sets from the HuGeneFL array and the 218 genes that are up-regulated in the tamoxifen-resistant down-regulated probe sets from the Hu95Av2 and 67 compared with the tamoxifen-sensitive breast cancer down-regulated probe sets from the HuGeneFL array, xenograft model MaCa 3366, a set of genes was discovered respectively, were subjected to a BLAST analysis and that was annotated as IFN-inducible (Table 2A and B; Figs. annotation by Gene Ontology (GO) and OMIM. Nonre- 4A and 5A, and data not shown) such as IFITM2 (34-fold), dundant genes (n = 109) were regulated >2-fold in MaCa G1P2 (12-fold), G1P3 (14-fold), IFI44 (3.3-fold), IFITM1 3366/TAM as compared with MaCa 3366. The expression (2.9-fold), IFIT1 (3.5-fold), MX1 (5.2-fold), NMI (2.9-fold), values of differentially expressed nonredundant genes ISGF3G (2.4-fold), and PKRK (1.9-fold) that were all present on both array types or present on only one array, increased. The gene IFI27 was found to be up-regulated but regulated with a fold change >2, were ranked and the 11-fold in MaCa 3366/TAM treated permanently with genes showing the strongest differential expression in the tamoxifen compared with the MaCa 3366 model, but it was tamoxifen-resistant compared with the tamoxifen-sensitive not up-regulated in MaCa 3366/TAM untreated or treated human breast cancer xenograft model are displayed (Table with tamoxifen only short-term. Numerous genes were 2A and B). If more than one probe set was given for a identified which are down-regulated by short-term tamox- particular gene, then the probe sets with the highest fold ifen treatment of MaCa 3366 and are also down-regulated change determined the rank on the list. Differences in the in MaCa 3366/TAM (Table 2B; Figs. 4B and 5B, and data expression values can be explained on the one hand by notshown).Membersofthisclusterofgeneswith different tumor growth characteristics in the xenograft mice decreased expression are cyclin D1 (2.1- to 2.3-fold depend- from the two independent animal experiments which ing on the probe set), GATA3 (1.9-fold), PDZK1 (4.7-fold), should, however, be leveled out by pooling five animals IGFBP5 (8.4- to 10.3-fold depending on the probe set), gap per treatment group and, on the other hand, by the fact that junction protein 43 [GJA1, gap junction protein a1, from array generation HuGeneFL to Hu95Av2 the number connexin 43 (3.8-fold)], and the homeobox transcription of probe sets on the array was increased from 6,574 to factors MSX1 and MSX2 (2.4- and 3.2-fold, respectively). 12,386 explaining why some genes are only present on the Among the genes decreased in tamoxifen-resistant xeno- Hu95Av2 array. grafts, either untreated or short-term tamoxifen treated, are Functional Categories of Differentially Expressed also the estrogen-related receptor g (ESRRG) and insulin- Genes like growth factor 1 receptor (IGF1R; Fig. 5B). For the 109 nonredundant genes differentially expressed Several genes that are associated with breast cancer are and listed in Table 2A and B, either a known function or differentially expressed, [e.g., breast carcinoma amplified an inferred function was identified from OMIM and GO sequence 1 (BCAS1) was up-regulated 4.1-fold in MaCa nomenclature (42, 43). GO stores controlled vocabulary 3366/TAM under permanent tamoxifen treatment com- organized under the three independent ontologies ‘‘bio- pared with untreated MaCa 3366]. The UDP-glycosyltrans- logical process,’’ ‘‘molecular function,’’ and ‘‘cellular ferase 2 family, member B15 (UGT2B15) is increased 3.2- to component.’’ The differentially expressed genes were 7.9-fold depending on the probe set in MaCa 3366/TAM categorized according to these ontologies (Fig. 4). In under permanent tamoxifen treatment compared with addition, we introduced three categories reflecting the MaCa 3366. In addition, many genes that had not biological system (i.e., ‘‘known association with cancer’’, previously been implicated in tamoxifen resistance were ‘‘IFN-inducible genes’’ (Table 2A), and ‘‘estrogen-respon- also identified: among the genes sharply up-regulated on sive genes’’ (Table 2A and B). As all three GOs can be both array types in the tamoxifen-resistant xenograft MaCa attributes of a particular gene, they can be assigned 3366/TAM, either untreated or short-term tamoxifen independently to it. In the GO biological process, molecular treated, are bone marrow stem cell antigen-2 (BST2) and function, and cellular component, the distribution of genes neuronatin (NNAT). to the categories was very similar for genes that are up- or Validation of Array Data with Quantitative Reverse down-regulated in MaCa 3366/TAM (Fig 4A and B) with Transcription-PCR. To confirm the differential gene three exceptions: the immune/defense response group, the expression from the array experiments employing an IFN-inducible genes, and the lipid metabolism group. independent method, we did real-time quantitative reverse Numerous IFN-inducible genes were found that were transcription-PCR in xenograft RNA samples (untreated coordinately up-regulated in MaCa 3366/TAM (Table 2A; and short-term tamoxifen treated MaCa 3366, untreated, Fig. 4A). Similarly, numerous estrogen-responsive genes short-term, and permanent tamoxifen-treated MaCa 33666/ were down-regulated in MaCa 3366/TAM (Table 2B; TAM) pooled from five animals each and from two Fig. 4B). Examples for up- and down-regulated genes are independent animal experiments. We selected four genes displayed in Fig. 5. The graphs show the high degree of with strong differential expression (NNAT, BST2, BCAS1,

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Figure 4. Gene Ontology categories. The 54 genes that are up-regulated (A) and the 55 genes that are down-regulated (B)in MaCa 3366/TAM were categorized according to involvement in biological processes, molecular function, and according to cellular component. The GO biological process is divided into immune/defense response, development/morphogenesis, cell growth, cell signaling, metabolism, and apoptosis. The GO molecular function is divided into signal transduction, transcription, protein synthe- sis/modification/transport, and lipid metabolism. In addition, we introduced three categories reflecting the biological system (i.e., IFN-inducible genes, known association with cancer, and estrogen-responsive genes). The GO category cellular component is divided into the four components: membrane, nucleus, intracellular, and extracellular. One gene can be a member of more than one GO category.

and IGFBP5) and two genes with a weaker differential Hu95Av2 array (2.2-fold) was confirmed with the Taqman expression (ESRRG and PTK6). The normalized gene assay (2-fold). However, ESRRG was not decreased in RNA expression levels relative to the expression in untreated from the first xenograft experiment as tested by Taqman MaCa 3366 as calibrator are shown in Fig. 6. TaqMan PCR (data not shown). The increase (2.4 on Hu95Av2 array) analysis clearly confirmed the increase of NNAT, BST2, of the breast cancer-related tyrosine kinase PTK6/Brk in and BCAS1 and the decrease of IGFBP5 in MaCa 3366/ MaCa 3366/TAM compared with MaCa 3366 was not TAM (Fig. 6). NNAT and BST2 were sharply up-regulated confirmed by Taqman PCR (data not shown). in MaCa 3366/TAM under permanent tamoxifen treatment compared with untreated MaCa3366 as measured by TaqMan analysis in RNA derived from the first and second Discussion xenograft experiment. The down-regulation of ESRRG in In the past, numerous estrogen-responsive genes MaCa 3366/TAM compared with MaCa 3366 on the were identified. Many of them are down-regulated after

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Figure 5. Affymetrix expression profile of selected genes. The values are derived from probe sets on the Hu95Av2 array (black and dark gray) and/or HuGeneFL array (light gray; see Table 2A and B for reference). The gene symbol here is the OMIM abbreviation, if available. A, examples of genes up- regulated in MaCa 3366/TAM compared with MaCa 3366 are neuronatin (NNAT), bone marrow stromal cell antigen 2 (BST2), UDP-glycosyltransferase 2 family, member B15 (UGT2B15); breast carcinoma amplified sequence 1 (BCAS1); myxovirus resistance 1 (MX1); IFN-induced gene with tetratricopeptide repeats 1 (IFIT1); IFN- induced transmembrane protein 2 (IFITM2); and IFN a – inducible protein 27 (IFI27). B, examples of genes down- regulated in MaCa 3366/TAM compared with MaCa 3366 are insulin-like growth factor binding protein 5 (IGFBP5),PDZdomaincontaining1 (PDZK1), estrogen-related receptor g (ESRRG), and insulin-like growth factor 1 receptor (IGF1R).

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Table 2. Summary of the differentially regulated genes in the human breast cancer xenograft model MaCa 3366/TAM compared with MaCa 3366

A. Genes that are reproducibly up-regulated in untreated or permanently tamoxifen-treated MaCa 3366/TAM compared with MaCa 3366

Fold increase in Fold increase in untreated MaCa MaCa 3366/TAM 3366/TAM plus permanent Gene Description Affymetrix probe ID GO* Reference versus untreated tamoxifen treatment symbol MaCa 3366 versus untreated MaCa 3366

HuGeneFL Hu95Av2 HuGeneFL Hu95Av2 HuGeneFL Hu95Av2

BST2 Bone marrow stromal cell antigen 2 24 37.9 39061_at 4.1 11 D28137_at NNAT Neuronatin 14 12.6 39051_at 1.4 2.6 U25034_s_at IFITM2 IFN-induced transmembrane 11 34 X57351_s_at i (44) protein 2 (1 – 8 D) G1P2 IFN-stimulated protein, 15 kDa 6.3 12 1107_s_at i (45) 2.0 3.6 38432_at G1P3 IFN, a-inducible protein 4.5 14 1358_s_at i (45) (clone IFI-6-16) 1.2 3.3 U22970_rna1_s_at GAGE8 G antigen 1 4.0 2.6 31497_at c (42) CRABP1 Cellular retinoic acid binding 3.9 1.0 542_at protein 1 2.8 1.2 543_g_at 2.1 1.2 S74445_at ACTA1 Actin, a 1, skeletal muscle 3.5 2.0 31737_at 2.1 1.5 M20543_at PPP2R2A Protein phosphatase 2 (formerly 2A), 3.2 1.0 41167_at regulatory subunit B, a isofom 1.7 1.5 M64929_at TAGLN Transgelin 3.0 2.1 36931_at 1.9 1.9 M95787_at B2M h-2-microglobulin 2.9 6.2 428_s_at 2.3 5.1 201_s_at 1.8 3.9 34644_at 2.6 6.1 J00105_s_at 2.0 3.4 S82297_at TM4SF8 Tetraspan 3 2.8 3.3 38612_at 2.9 4.9 M69023_at BCAS1 Breast carcinoma amplified 2.8 4.1 37821_at c (42) sequence 1 CPE Carboxypeptidase E 2.7 4.4 36606_at 2.1 7.9 X51405_at ID1 Inhibitor of DNA binding 1 2.6 3.0 36617_at SEPP1 Selenoprotein P, plasma, 1 2.5 3.6 34363_at 2.4 6.0 Z11793_at ID3 Inhibitor of DNA binding 3 2.5 2.8 37043_at PTK6 Protein tyrosine kinase 6 2.4 3.0 33559_at c (42) NMI N-myc (and STAT) interactor 2.4 2.9 36472_at i, c (42, 46) 1.5 3.2 U32849_at CACNB3 Calcium channel, voltage- 2.3 3.0 34726_at dependent, h 3 submit 1.5 2.1 U07139_at RNASE4 Ribonuclease, RNase A family, 4 2.2 3.2 32664_at 2.2 5.2 D37931_at E2F1 E2F transcription factor 1 2.2 2.5 2028_s_at c (42) PROCR Protein C receptor, endothelial 1.9 2.2 647_at (EPCR) 2.1 2.5 L35545_at NMT1 N-myristoyltransferase 1 1.9 2.1 38999_s_at 1.6 1.5 M86707_at

NOTE: The expression values of differentially expressed non-redundant genes were ranked for the genes up- and down-regulated, respectively, in the tamoxifen-resistant compared to the tamoxifen-sensitive human breast cancer xenograft model. The gene symbol here is the most commonly used abbreviation, usually from OMIM, if available. Affymetrix probe ID is the unique name of the probe set on the HuGeneFL or Hu95Av2 array. Estrogen- responsive and IFN-inducible genes as well as genes associated with cancer are indicated. * i, IFN-inducible; e, estrogen-responsive; c, known association with cancer.

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Table 2. Summary of the differentially regulated genes in the human breast cancer xenograft model MaCa 3366/TAM compared with MaCa 3366 (Cont’d)

A. Genes that are reproducibly up-regulated in untreated or permanently tamoxifen-treated MaCa 3366/TAM compared with MaCa 3366

HuGeneFL Hu95Av2 HuGeneFL Hu95Av2 HuGeneFL Hu95Av2

ISGF3G IFN-stimulated transcription 1.9 2.4 38517_at i (47) factor 3, g (48 kDa) 2.0 4.9 M87503_at IFITM1 IFN-induced transmembrane 1.9 2.9 676_g_at i (48) protein 1 (9-27) LGALS3BP Lectin, galactoside-binding, soluble, 1.9 2.9 37754_at c (42) 3 binding protein 2.5 3.5 L13210_at GTF2E2 General transcription factor IIE, 1.9 1.6 37295_at polypeptide 2 1.9 1.6 X63469_at UGT2B15 UDP-glycosyltransferase 2 family, 1.9 4.1 33069_f_at c (42) polypeptide B15 1.4 3.2 33068_f_at 1.3 7.9 U08854_s_at BTG3 BTG family, member 3 1.8 1.9 37218_at 2.0 1.7 D64110_at SPS2 Selenophosphate synthetase 2 1.8 1.9 39078_at 1.5 1.7 U43286_at PITX1 Paired-like homeodomain 1.8 2.7 37920_at transcription factor 1 DECR1 2,4-Dienoyl CoA reductase 1, 1.8 1.8 38104_at mitochondrial 3.1 3.1 U49352_at IFI44 IFN-induced protein 44 1.7 3.3 37641_at i (49) 1.0 2.7 D28915_at EPHX1 Epoxide hydrolase 1, microsomal 1.7 1.7 38790_at 3.0 3.6 L25878_s_at 2.1 2.6 HG2465-HT4871_at SNX1 Sorting nexin 1 1.6 1.7 36583_at 2.6 2.8 U53225_at ARL4 ADP-ribosylation factor-like 4 1.6 1.5 33796_at 1.5 1.9 U73960_at N33 Putative prostate cancer 1.5 2.0 36852_at c (42) tumor suppressor 1.6 3.5 U42359_at 2.4 3.3 U42360_cds2_at EBAG9 Estrogen receptor binding site 1.5 1.6 38283_at e, c (42, 50) associated, antigen, 9 CXADR Coxsackie virus and adenovirus 1.5 1.8 37534_at receptor 1.5 2.2 U90716_at IFIT1 IFN-induced protein with 1.4 3.5 32814_at i (51) tetratricopeptide repeats 1 1.0 5.6 M24594_at SCGB2A2 Secretoglobin, family 2A, member 2 0.7 2.3 36329_at EGR3 Early growth response 3 4.4 1.0 X63741_s_at TFF3 Trefoil factor 3 (intestinal) 3.0 3.1 L08044_s_at NRIP1 Nuclear receptor interacting protein 1 2.8 1.1 X84373_at e (52) TFPI2 Tissue factor pathway inhibitor 2 2.6 1.9 L27624_s_at 2.3 2.5 D29992_at TFF1 Trefoil factor 1 2.3 0.4 X52003_at e, c (42, 53) CTSB Cathepsin B 2.3 3.1 HG417-HT417_s_at c (42) CTSD Cathepsin D 2.2 1.4 M63138_at e, c (42, 54, 55) PRKR IFN-inducible RNA-dependent 1.3 1.9 U50648_s_at i (56) protein kinase TM4SF3 Transmembrane 4 superfamily 1.2 5.9 M35252_at c (42) member 3 IRF2 IFN regulatory factor 1.2 2.0 X15949_at MX1 Myxovirus resistance 1, 1.0 5.2 M33882_at i (57) IFNa-inducible protein p78 IFI27 IFN, a-inducible protein 27 1.0 11 X67325_at i (58)

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Table 2. Summary of the differentially regulated genes in the human breast cancer xenograft model MaCa 3366/TAM compared with MaCa 3366 (Cont’d)

B. Genes that are reproducibly down-regulated in untreated or permanently tamoxifen-treated MaCa 3366/TAM compared with untreated MaCa 3366 xenograft tumors

Fold decrease in Fold decrease in untreated MaCa MaCa 3366/TAM 3366/TAM plus permanent Gene Description Affymetrix probe ID GO* Reference versus untreated tamoxifen treatment symbol MaCa 3366 versus untreated MaCa 3366

HuGeneFL Hu95Av2 HuGeneFL Hu95Av2 HuGeneFL Hu95Av2

ELOV2 Elongation of very long chain 9.9 11 39642_at fatty acids-like 2 KHDRBS3 KH domain containing, RNA 6.5 7.5 31786_at binding, signal transduction associated 3 LRP2 Low density 5.9 5.9 35009_at lipoprotein-related protein 2 PDZK1 PDZ domain containing 1 4.7 2.9 32027_at e (59) MSX2 Msh homeo box homologue 2 4.6 3.2 40734_r_at e (60, 61) (Drasophila) PEG10 Paternally expressed 10 4.0 4.5 39696_at c (42) ALDH3B2 Aldehyde dehydrogenase 3 3.5 3.5 37956_at family, member B2 1.8 1.4 U37519_at TCN1 Transcobalamin I 3.2 6.5 35919_at (vitamin B12 binding protein) IGFBP5 Insulin-like growth factor 3.1 10 1396_at e (62, 63) binding protein 5 2.7 8.4 38650_at 3.1 5.0 L27560_at 1.5 1.5 L27559_s_at CLCA2 Chloride channel, Calcium 3.0 3.0 40356_at c (42) activated, family member 2 GJA1 Gap junction protein, a 1, 43 kDa 3.0 3.7 32531_at e (64) (connexin 43) 1.0 5.6 X52947_at KAB1 KARP-1 binding protein, KIAA0470 2.9 2.4 33893_r_at gene product C14orf147 Hypothetical protein MGC24447 2.8 2.1 33400_r_at TRG T-cell receptor g locus 2.8 2.8 41468_at 3.3 2.8 M30894_at FGFR1 Fibroblast growth factor receptor 1 2.7 2.7 424_s_at c (42) (fms-related tyrosine kinase 2) 2.7 3.4 2057_g_at 1.6 2.5 2056_at 0.8 3.2 X66945_at DUSP6 Dual specificity phosphatase 6 2.7 2.5 41193_at MGC13523 Malic enzyme 1, NADP(+)- 2.7 2.1 31824_at dependent, cytosolic 2.3 1.7 U43944_at TTK TTK protein kinase 2.6 2.3 572_at 2.4 2.5 M86699_at IL8 Interleukin 8 2.5 1.3 35372_r_at 2.0 2.4 Y00787_s_at OSBPL1A Oxysterol binding protein-like 1A 2.5 3.5 36689_at DKFZp564H1916 Homo sapiens cDNA DKFZp564H1916 2.4 2.0 41229_at PDCD4 Programmed cell death 4 2.4 2.5 39510_r_at (neoplastic transformation inhibitor) QKI Homologue of mouse quaking QKI 2.4 2.7 39760_at (KH domain RNA binding protein) IL1R1 Interleukin 1 receptor, type I 2.4 2.8 1368_at 1.6 1.0 M27492_at CCND1 Cyclin D1 2.3 2.3 2017_s_at e, c (42, 65) 2.0 2.1 38418_at ESRRG Estrogen-related receptor g 2.2 2.2 31843_at TM4SF1 Transmembrane 4 superfamily 2.2 1.7 892_at c (42) member 1 2.2 1.5 41531_at

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Table 2. Summary of the differentially regulated genes in the human breast cancer xenograft model MaCa 3366/TAM compared with MaCa 3366 (Cont’d)

B. Genes that are reproducibly down-regulated in untreated or permanently tamoxifen-treated MaCa 3366/TAM compared with untreated MaCa 3366 xenograft tumors

HuGeneFL Hu95Av2 HuGeneFL Hu95Av2 HuGeneFL Hu95Av2

SEMA3C Semaphorin 3C 2.2 2.0 377_g_at c (42) ZNF43 Zinc finger protein 43 (HTF6) 2.1 2.0 33982_f_at RBP1 Retinol binding protein 1, cellular 2.1 2.1 38634_at c (42) 2.4 2.4 X07438_s_at GATA3 GATA binding protein 3 2.0 1.9 40511_at e (66) IRF3 IFN regulatory factor 3 2.0 2.0 371_at AIM1 Absent in melanoma 1 2.0 2.3 32112_s_at c (42) 1.7 1.8 U83115_at MSX1 Msh homeo box homologue 1 2.0 2.4 40199_at e (60) ME1 Malic enzyme 1, NADP(+)- 1.9 2.2 837_s_at dependent, cytosolic LBR Lamin B receptor 1.9 1.6 288_s_at 1.5 2.5 L25931_s_at BCAS2 Breast carcinoma amplified 1.9 1.7 34286_at c (42) sequence 2 PKIA Protein kinase (cAMP-dependent, 1.9 2.2 546_at catalytic) inhibitor a 1.7 2.2 36202_at 1.5 1.4 S76965_at #23612 Human clone 23612 mRNA sequence 1.8 1.8 38370_at 2.6 2.6 U90902_at EMP1 Epithelial membrane protein 1 1.8 1.6 37762_at LENG4 Leukocyte receptor cluster 1.8 1.8 180_at c (42) (LRC) member 4 2.0 1.9 181_g_at 1.4 1.3 S82470_at AGPS Alkylglycerone phosphate synthase 1.8 1.8 39225_at 1.6 4.5 Y09443_at HDAC2 Histone deacetylase 2 1.8 1.9 34368_at 1.6 2.1 U31814_at IGF1R Insulin-like growth factor 1 receptor a chain 1.7 6.4 34718_at c (42) COX7A2 Cytochrome c oxidase subunit 1.7 1.8 41760_at VIIa polypeptide 2 (liver) 1.7 1.7 X15822_at TM4SF6 Transmembrane 4 superfamily member 6 1.7 1.4 39362_r_at ORC3L Origin recognition complex, subunit 3-like 1.7 1.7 36895_at 2.8 2.2 U50950_at CD24 CD24 antigen 1.6 1.8 266_s_at c (42) 1.6 1.1 L33930_s_at ADAM17 A disintegrin and metalloproteinase domain 17 1.6 1.6 41601_at 1.7 1.4 U69611_at PTPRK Protein tyrosine phosphatase, receptor type, K 1.6 1.8 1488_at c (42) 1.6 1.6 L77886_at ACAA2 Acetyl-coenzyme A acyltransferase 2 1.4 1.8 41530_at (mitochondrial 3-oxoacyl- 1.8 2.0 D16294_at coenzyme A thiolase) AREG Amphiregulin 1.3 12 34898_at e, c (42, 67) 1.3 4.4 M30703_s_at TRIM16 Tripartite motif-containing 16 1.2 2.1 38881_i_at e (68) LIPF Lipase, gastric 2.5 2.5 X05997_at NR2F2 Nuclear receptor subfamily 2, group F, 1.5 1.5 M64497_at member 2

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Figure 6. Representation of TaqMan-PCR results. Columns, mean values for each cDNA derived from pooled RNAs from five animals; bars, FSD. Each real-time TaqMan PCR was done in triplicate. The amount of a given mRNA was first normalized to the level of the housekeeping gene HMBS. Fold differences were then computed using normalized values (standard curve method). The fold change is expressed relative to the amount of mRNA present in the calibrator MaCa 3366. Neuronatin (NNAT), bone marrow stromal cell antigen 2 (BST2), and BCAS1 are up-regulated in MaCa 3366/TAM. The same expression value of BCAS1 was measured for untreated MaCa 3366/TAM as in the calibrator in the RNA sample from the first xenograft experiment. Insulin-like growth factor binding protein 5 (IGFBP5) is down-regulated in MaCa 3366/TAM. Experiments with RNA from the first (gray columns) and second (black columns) xenograft experiments.

short-term tamoxifen treatment and after permanent By real-time PCR, the strong differential expression was tamoxifen treatment in the tamoxifen-resistant breast confirmed (Fig. 6). NNAT is highly expressed in the brain of cancer xenograft MaCa 3366/TAM. Members of this group the murine fetus and neonate and at much lower levels in the of genes which previously were shown to be estrogen- brain of the adult (69). The function of NNAT is unknown, but responsive and which are down-regulated in MaCa 3366/ its functioning as a regulator of ion channels during brain TAM are cyclin D1 (65), GATA3 (66), PDZK1 (59), gap development has been proposed (70). Thus far, no association junction protein 1 GJA1 (64), and the homeobox transcrip- of NNAT expression with breast cancer and tamoxifen- tion factors MSX1 and MSX2 (60). MSX2 was shown to be resistant breast cancer in particular has been shown. induced by E2 in the human breast cancer cell line MCF-7 Breast carcinoma amplified sequence 1 (BCAS1) was up- (61). The growth factor amphiregulin, which modulates regulated (4.1-fold on the Hu95Av2 array) in MaCa 3366/ growth by binding to the epidermal growth factor receptor, TAM treated permanently with tamoxifen compared with was sharply down-regulated in MaCa 3366/TAM. It is well MaCa 3366 (Table 2A). By real-time PCR, the up-regulation known that estrogen induces the expression of amphir- was confirmed (Fig. 6). BCAS1 also known as novel egulin in human breast carcinoma cells and that 4-OHT amplified in breast cancer-1 (NABC1) lies in 20q13.3, a interferes with the induction of amphiregulin by estrogen region often amplified in breast carcinoma, and is in cell lines (67). Down-regulation of IGFBP5 (62, 63) was associated with aggressive tumor behavior. BCAS1 was confirmed by real-time PCR (Fig. 6). The IGF1R was down- found to be overexpressed in breast carcinomas and certain regulated in MaCa 3366/TAM. In a comparative study breast cancer cell lines on the RNA and protein level of different antiestrogen-resistant cell lines, reduced (71, 72). Surprisingly, BCAS1 mRNA is expressed at high levels of IGF1R were detected and it was proposed that levels only in normal brain and prostate (72). Currently, the this protein might be a general marker for antiestrogen function of BCAS1 is unknown; however, overexpression of resistance (14). human BCAS1 in NIH-3T3 cells did not affect the growth NNAT was found to be sharply up-regulated in MaCa rate or anchorage-independent growth suggesting that 3366/TAM compared with MaCa 3366 (Table 2A; Fig. 5). BCAS1 is not a typical proto-oncogene (72).

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Metabolism of steroid hormones are subject to glucur- cancer cells (58), was up-regulated 11-fold in MaCa 3366/ onidation which is a major pathway for the elimination of TAM treated permanently with tamoxifen compared with steroids in liver and several other steroid target tissues. MaCa 3366 but it was not up-regulated in untreated or Glucuronidation is catalyzed by UDP-glycosyltransferases, short-term tamoxifen treated MaCa 3366/TAM (Fig. 5). which transfer glucuronic acid to a wide variety of In the tamoxifen-resistant breast cancer xenograft model endogenous and exogenous compounds. The UDP-glycosyl- the bone marrow stromal cell antigen 2 (BST2) was sharply transferase 2 family, member B15 (UGT2B15)isup- up-regulated and differential expression was confirmed by regulated 3.2- to 7.9-fold depending on the probe set in real-time PCR (Fig. 6). BST2 is also known as HM1.24 MaCa 3366/TAM under permanent tamoxifen treatment antigen and is a type II membrane glycoprotein that is compared with MaCa 3366 (Table 2A; Fig. 5). It was shown preferentially expressed on terminally differentiated B that recombinant UGT2B15 selectively glucuronidates cis- cells (80). The promoter contains three STAT3 DNA- 4-OHT, the active metabolite of tamoxifen (73). It is of binding sites (81). Expression of BST2 might therefore interest that cis-4-OHT existed at a higher concentration directly be induced by STAT3. A human anti-BST2 than trans-4-OHT in a MCF-7 breast carcinoma xenograft antibody was generated which is currently being tested model of tamoxifen-administered mice who had become for the immunotherapy of multiple myeloma (82). Recently, resistant to tamoxifen in the course of tamoxifen treatment it has been shown that the rat homologue of BST2/HM1.24 (74). In addition, it was shown that acquired resistance to has both a cell surface and an intracellular localization and tamoxifen is associated with markedly reduced cellular it is efficiently internalized from the cell surface (83). levels of tamoxifen together with an accumulation of The fact that IFN-inducible genes are up-regulated in the less antiestrogenic cis-4-OHT in human breast cancer xenograft tumors is not unprecedented. When human patients who had become resistant to tamoxifen (75). breast tumors were analyzed by gene expression profiling, Possibly, tamoxifen administration induces the expression a set of coexpressed genes was identified which was annotated as being IFN-stimulated (20, 21). The strength of of UGT2B15, which metabolizes and inactivates cis-4-OHT. expression of these genes correlated positively with the Interestingly, the phenol sulfotransferase SULT1A that was protein expression of STAT1 and the authors suggested shown to sulfate 4-OHT (73) was induced by tamoxifen in that high levels of STAT1 and STAT3 could increase the the human breast cancer cell line ZR-75-1, as shown by expression of a set of IFN-inducible genes (20). Moreover, SAGE analysis (32), suggesting a role for tamoxifen in a gene expression profiling analysis of ductal carcinoma metabolizing enzymes in the development of tamoxifen in situ as compared with invasive ductal carcinoma (84), resistance. IFN-inducible genes were most strongly up-regulated in Many IFN-inducible genes were up-regulated in MaCa the ductal carcinoma in situ phase of the breast carcinomas. 3366/TAM as compared with the MaCa 3366 xenograft Although we did not identify STAT1 and STAT3 as (Table 2A). Via activation of the JAK signal transducer and transcriptionally up-regulated genes in MaCa 3366/TAM activator of transcription (STAT) pathway, IFNs but also by Affymetrix array analysis, it is conceivable that they are other cytokines like IL-6 elicit multiple biological post-transcriptionally activated (e.g., by growth factor responses, which are mediated by the proteins encoded receptor or receptor tyrosine kinase mediated phosphory- by IFN-inducible genes. Phosphorylated STAT proteins lation). Additionally, it is well known that STAT3 and to bind DNA sequences in the promoters of IFN-inducible some extent also STAT1 can be activated in breast genes that are called g-IFN activated sequences and IFN- carcinomas (85) and that constitutively active mutants of stimulated response elements (76). Two IFN-inducible STAT3 are oncogenic (86–89). Activated STAT3 promotes genes, the double-stranded RNA-activated protein kinase the growth and survival of tumor cells thereby contributing PRKR and MX1, which belongs to the dynamin family of to malignancy (86, 89). STAT1 and STAT3 can be activated large GTPases (77), had an increased expression in MaCa by various receptor and non-receptor tyrosine kinases (e.g., 3366/TAM compared with MaCa 3366 (Tables 2A and 3, the epidermal growth factor receptor and c-Src; refs. 86, 88). Fig. 5). Interestingly, MX1 was previously identified in a Moreover, it was shown in vitro that E2-mediated inhibi- comparative genomic and proteomic analysis of the tion of IL-6–induced STAT3 activation was reversed by human breast cancer cell line T47D and its antiestrogen tamoxifen indicating that under permanent tamoxifen (Faslodex , ZM 182780)-resistant derivative T47D-r (39, 78). treatment STAT3 could indeed be hyperactivated (90, 91) In this study, MX1 was sharply up-regulated in the One may assume that heterodimers of the transcription antiestrogen-resistant cell line T47D-r on the mRNA and factors STAT1/STAT2 and STAT11/STAT3, which bind to protein level. IFN-stimulated response elements (76) are involved in the The biological functions of many other IFN-inducible up-regulation of MX1 and BST2. Thus, the up-regulation genes that were coordinately up-regulated in the tamoxi- seen for MX1 and BST2 in MaCa 3366/TAM could be fen-resistant as compared with the parental xenograft explained by an activated STAT pathway in these cells. tumor such as IFITM1, IFITM2, GIP2 and GIP3 (45), Activation of STAT3 in MaCa 3366/TAM might be IFIT1 (51), and IFI44 (49) is still completely unknown (79). responsible for the up-regulation of the numerous IFN- In contrast to the other IFN-inducible genes, IFI27, which inducible genes that contain an IFN-stimulated response was initially identified from E2-treated human breast element in their promoter.

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In conclusion, this study provides a detailed analysis of characterization of a tamoxifen-resistant breast carcinoma xenograft. Br J Cancer 2000;82:1844 – 50. the genes that are up- and down-regulated in an in vivo 11. Fichtner I, Becker M, Zeisig R, Sommer A. In vivo models for model of tamoxifen resistance. More than 100 transcripts endocrine-dependent breast carcinomas: special considerations of clinical are changed in abundance in MaCa 3366/TAM as relevance. Eur J Cancer 2004;40:845 – 51. compared with MaCa 3366. Our future studies will aim at 12. Clarke R, Skaar TC, Bouker KB, et al. Molecular and pharmacological aspects of antiestrogen resistance. J Steroid Biochem Mol Biol identifying which of the genes in the tamoxifen-resistant 2001;76:71 – 84. xenograft model contribute most to the phenotype. Among 13. Katzenellenbogen BS, Montano MM, Ekena K, Herman ME, the genes that are differentially expressed in the tamoxifen- McInerney EM. William L. McGuire Memorial Lecture. 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