JOURNAL OF CELLULAR PHYSIOLOGY 200:440–450 (2004) Molecular Identification of ERa-Positive Breast Cancer Cells by the Expression Profile of an Intrinsic Set of Estrogen Regulated Genes ALESSANDRO WEISZ,1* WALTER BASILE,1 CLAUDIO SCAFOGLIO,1 LUCIA ALTUCCI,1 FRANCESCO BRESCIANI,1 ANGELO FACCHIANO,2 PIERO SISMONDI,3,4 LUIGI CICATIELLO,1 3,5 AND MICHELE DE BORTOLI 1Dipartimento di Patologia Generale, Seconda Universita` Degli Studi di Napoli, Vico L. De Crecchio 7, Napoli, Italy 2Istituto di Scienze dell’Alimentazione del Consiglio Nazionale delle Ricerche, Avellino, Italy 3IRCC-Institute for Cancer Research & Treatment, University of Turin, Candiolo (TO), Italy 4Department of Gynecology & Obstetrics, University of Turin, Candiolo (TO), Italy 5Department of Oncological Sciences, University of Turin, Candiolo (TO), Italy Estrogens exert a key biological role in mammary gland epithelial cells and promote breast carcinogenesis and tumor progression. We recently identified a new large set of estrogen responsive genes from breast cancer (BC) cells by DNA microarray analysis of the gene expression profiles induced by 17b-estradiol in ZR-75.1 and MCF-7 cells. The purpose of the present study was to test whether the expression pattern of hormone regulated genes from this set identifies estrogen receptor (ERa) positive, hormone responsive BC cells. To this aim, we carried out in silico metanalysis of ERa positive and ERa negative human BC cell line transcriptomes, focusing on two sets of 171 and 218 estrogen responsive genes, respectively. Results show that estrogen dependent gene activity in hormone responsive BC cells is significantly different from that of non-responsive cells and, alone, allows to discriminate these two cellular phenotypes. Indeed, we have identified 61 genes whose expression profile specifically marks ERa positive BC cells, suggesting that this gene set may be exploited for phenotypic characterization of breast tumors. This possibility was tested with data obtained by gene expression profiling of BC surgical samples, where the ERa positive phenotypes were highlighted by the expression profile of a subset of 27 such hormone responsive genes and four additional BC marker genes, not including ERs. These results provide direct evidence that the expression pattern of a limited number of estrogen responsive genes can be exploited to assess the estrogen signaling status of BC cells both in vitro and ex-vivo. J. Cell. Physiol. 200: 440–450, 2004. ß 2004 Wiley-Liss, Inc. Breast cancer (BC) remains a serious threat to the lump. Since no marker is available today to distinguish patients, despite significant improvements in its clinical cancers most likely to recur, there is today a tendency to management (Harris et al., 2000). The most informative prescribe adjuvant treatments to all patients. The kind prognostic factor for primary BC is the presence of of pharmacological treatment is based on clinico- lymph node metastases: around 70% of node-negative pathological criteria and on the expression of biological patients can be expected to survive BC without markers in tumor cells, since lymph-node metastasis per additional treatments beyond surgical resection of the se is not predictive of sensitivity or resistance of the Contract grant sponsor: Italian Association for Cancer Research (to AW and PS); Contract grant numbers: IG 2002, IG 2003; Contract grant sponsor: Italian Ministry for Education, Univer- sity and Research (PRIN 2002-03 and FIRB Post-genoma Grant to AW and MDB); Contract grant numbers: 2002067514_002, RBNE0157EH; Contract grant sponsor: European Commission (contracts to AW and LA); Contract grant numbers: BMH4-CT98- *Correspondence to: Alessandro Weisz, Dipartimento di Patologia 3433, QLG1-CT-2000-01935, QLK3-CT-2002-02029; Contract Generale, Seconda Universita` degli Studi di Napoli, Vico L. De grant sponsor: Ministry of Health; Progetti Speciali 2000 and Crecchio, 7, 80138 Napoli, Italy. 2002 (to AW, MDB, and PS); Contract grant sponsor: Second E-mail: [email protected] University of Naples; Ricerca di Ateneo 2002 and 2003 (to AW, FB, and LA); Contract grant sponsor: Regione Piemonte, Ricerca Received 31 July 2003; Accepted 3 December 2003 Sanitaria Finalizzata 2002, 2003 (to PS). DOI: 10.1002/jcp.20039 ß 2004 WILEY-LISS, INC. MOLECULAR TYPING OF ERa POSITIVE BREAST CANCER CELLS 441 disease to any of the adjuvant treatments available 2002). It is not clear, however, whether such patterns (Lønning et al., 2001). are indeed related to the estrogen signaling status of the Adjuvant therapy is the most effective weapon tumor or, instead, if they reflect its differentiation available today against recurrence and systemic spread status. of BC. Pharmacological treatments, involving the use of To help address this problem, we speculated that the non-selective cytotoxic treatments and/or endocrine identification of a set of hormone responsive genes in therapy, reduce the recurrence rate and the risk of human BC cells could provide specific markers to distant metastases. Similar pharmacological treat- monitor the hormone signaling status of tumors. ments are also used in the advanced disease. Endocrine Expression profiling of a specific set of estrogen therapy, based on systemic estrogen ablation by drugs responsive genes, in fact, would allow focusing the affecting hormone synthesis, metabolisms or actions in analysis on the hormone dependent transcriptome of BC target cells, is very effective on selected patients and cells. We present here evidence that a subgroup of generally well tolerated, both in the adjuvant setting hormone-responsive genes is indeed sufficient to iden- and in the metastatic disease. Hormone responsive tify ER-positive BC cells, either in culture or from tumor breast tumors are currently identified by the presence of biopsies. This is proof of concept that hormone-depen- ERs (ERa and in some cases also ERb) and of the dent gene expression profiles can identify ER pathway estrogen regulated progesterone receptors (PgRs). Pre- activation in tumors and thus may be used to predict diction of BC responsiveness to hormones, based on hormone-responsiveness in BC. current assays for ERa expression in tumor cells can, however, be misled by the existence of different MATERIALS AND METHODS molecular situations in which the ER is present but Selection of the starting set of inactive (Ferguson et al., 1998; Hopp and Fuqua, 1998). estrogen-responsive genes Indeed, comprehensive clinical trials have shown that The starting set of estrogen responsive genes used for about one third of ER/PgR-positive tumors fail to all the analysis was derived from our microarray study respond to endocrine therapy, while about 10% of of the genomic response of human MCF-7 and ZR75.1 receptor-negative ones do respond (Harris et al., 2000). BC cell lines to estrogen treatment (Weisz et al., 1999; Receptor-positive tumors are thus heterogeneous with Cicatiello et al., 2001; Cicatiello L, Natoli G, Scafoglio C, respect to estrogen sensitivity and, for this reason, Altucci L, Cancemi M, Facchiano A, Calogero R, Iazzetti functional markers of hormone dependent signaling or G, De Bortoli M, Sfiligoi C, Sismondi P, Biglia N, ERs function in BC cells are being actively searched for Bresciani F, and Weisz A. The gene expression program (Biswas et al., 1998; Bouras et al., 2001; Finlin et al., activated by estrogen in hormone responsive human BC 2001). Identification of such markers is complicated, cells. Submitted for publication elsewhere). This set was however, by the fact that hormone receptors expression produced using cDNA microarrays containing probes for in BC cells more often is associated with cell differentia- 8,372 unique human genes/EST and allowed to identify tion and low proliferation rates. 344 genes whose expression is either increased or Gene expression profiling is a promising new way to decreased after estrogen stimulation. classify BC in clinically relevant subtypes according to molecular ‘signatures’ of tumor biopsies (Martin et al., Expression data extraction and evaluation 2000; Perou et al., 2000; Sørlie et al., 2001; West et al., The first set of data was derived from the NCI60 gene 2001; Bertucci et al., 2002; van’t Veer et al., 2002). To expression database for the molecular pharmacology of provide the logical framework required for interpreting cancer (Ross et al., 2000) that includes the data relative to the biological significance of the gene expression the expression of approximately 8,000 genes in 60 cell patterns observed in tumor samples, however, the lines of different tissue origin. Seven BC cell lines are identification of the transcriptional programs associated present in this database, 2 ERa positive and estrogen with BC cell responses to relevant physiological stimuli responsive (T-47D and MCF-7) and 5 ERa negative, is much sought after. Perou et al. (1999), for example, hormone unresponsive (BT-549, Hs578T, MDA-MB-231, found that clusters of coexpressed genes identified MDA-MB-435, and MDA-N, an ERBB2-transfected sub- through manipulations of mammary epithelial cells clone of MDA-MB-435 cells). For comparison, data in vitro, including stimulation with growth factors and relative to one normal breast tissue biopsy (normal cytokines, showed consistent patterns of variation in breast) and 16 human cell lines from brain (SF-268 and expression among BC specimens. Also, Ross et al. (2000), U251), colon (COLO205 and HCT-116), kidney (A498 by comparing the gene expression patterns of BC and 786-0), lung (A549 and NCI-H322), ovary (OVCAR-4 biopsies and breast and mesenchymal cell lines found and -8), and prostate (DU-145 and PC-3) cancer, specific gene expression features in tumors which melanoma (SKMEL-5and -28), chronic myeloid leukemia appeared to be related to what observed in vitro in cell (K-562), and multiple myeloma (RPMI-8226) were lines, where specific gene expression signatures could be considered. The microarray data relative to each of these linked to defined biological properties of the cells, such cell lines were downloaded from the NCI60 Cancer Micro- as doubling time, drug metabolism, or response to array Project web site (http://genome-www.stanford.edu/ interferon stimulation.