BMC Cancer 2008, 8:270 http://www.biomedcentral.com/1471-2407/8/270 Background Breast cancer comprises a series of distinct malignant tumors that present diverse cellular features with different stages and grades, distinct genetic changes, differing responses to therapy and varying outcomes [1]. Tailoring specific treatments to the disease's subtypes has tradition- ally been performed by histopathological analysis of tumor sections, supported by limited immunopathologi- cal and genetic assays [2]. Gene-expression profiling of human breast cancers has expanded our understanding of the clinical diversity of the disease and enabled a more accurate classification of tumors into subtypes, as well as a determination of their response to drug treatments [3,4]. The clinical benefits gained from profiling gene expres- sion in tumor biopsies have also provided better insight into the development and characteristics of the disease. For instance, the discovery of a unique set of genes that are predictive of metastases was associated with the recogni- tion that metastatic properties are determined in the pri- mary tumor relatively early in development. It also indicated that the molecular mechanism involved in bone marrow metastasis is different from that mediating lym- phatic spread [5-8]. Likewise, the "proliferation signature" encompasses a universal pattern of gene expression among tissues and predicts the outcome in patients [9]. It also implies that the regulation of some individual cell- cycle regulatory genes is more complex than simple restriction of transcription to certain phases of the cell cycle. Indeed, progress has been made in determining the molecular profiles of breast-tumor subtypes and their response to treatment [5,6,10,11]. However, the distinct contribution of defined metabolic pathways and their pri- mary elements to the overall phenotypic and functional diversity among tumors remains poorly understood. In breast cancer, rate of differentiation has been negatively correlated with the invasiveness and aggressiveness of the disease [12]. To elucidate the molecular and cellular char- acteristics and putative pathways that distinguish the poorly differentiated carcinoma and the highly differenti- ated papillary adenocarcinoma, global gene expression analysis was performed in transgenic mouse model. The tumors develop in about 8 to 9% of the aged, post-estro- pausal transgenic mice expressing variants of the signal transducer and activator of transcription 5 (Stat5) in their mammary glands. Both tumor types are of epithelial ori- gin, but strictly different in their pathological appearance. The carcinoma is an undifferentiated, non-glandular tumor, which can be distinguished by a typical structure of sheets of neoplastic mass with solid nests of poorly organized and discohesive cells. In contrast, the papillary adenocarcinoma is a highly differentiated tumor of glan- dular origin. Its microscopic structure is composed pre- dominantly of fibrovascular frond-like projections, covered by epithelial cells [13,14]. Divergent protein expression which is not associated with the type of trans- genic Stat5 variant expressed has been defined in these tumors, and a high degree of aneuploidy was demon- strated exclusively for the carcinoma epithelial cells [13]. Typically, cancer cells overexpress genes that are preferen- tially expressed in tissues other than those of the cancer's origin [15]. Thus, the apparent differences between these types of tumors could involve alterations in cellular and molecular functions, some of which are difficult to pre- dict. Global profiling of gene expression in these tumors has revealed the nucleus-cytoskeleton-plasma membrane axis as a target for altered gene expression which deter- mines the diversity among these tumors. Methods Mouse mammary-tumor samples Mammary tumors were developed in transgenic mice car- rying one of two Stat5 variants on a FVB/N background: (i) constitutively activated STAT5, termed STAT5ca, com- prising sequences from three genes: amino acids 1–750 from ovine Stat5, 677–847 from human Stat6, and 757–1129 from mouse Janus kinase (Jak) 2. and (ii) a deleted construct, STAT5Δ750, prepared by introducing a stop codon at the respective site of the native Stat5 DNA sequence, thus eliminating the expression of its transacti- vation domain. These constructs were inserted into the β- lactoglobulin (BLG) multiple-cloning site for mammary- gland-specific expression [16]. Upon identification, tumors were excised and snap-frozen for RNA isolation and validation studies. Biopsies were taken for histologi- cal analyses. All animals used in this study received humane care. The study protocols are in compliance with the regulation of the Israeli Ministry of Health and the local institutional policies (approval no. IL-39-03). Microarray hybridization and data analysis RNA was extracted from the individual tumors that were pathologically diagnosed [13], or from mammary glands with TRIZOL and reverse-transcribed. Each tumor or mammary gland was excised from different mouse. Equal amounts of cRNA from each tumor were hybridized to an Affymetrix GeneChip® Mouse Genome 430A 2.0 array (Affymetrix, Santa Clara, CA), which includes approxi- mately 14,000 annotated genes from the mouse genome. Hybridization and signal quantitation were performed according to Affymetrix's protocol by the Biological Serv- ices of the Weizmann Institute of Science (Rehovot, Israel). Briefly, 15 μg total RNA was reversed-transcribed using a T7-oligo(dT) promoter-primer in the first-strand DNA-synthesis reaction. Following RNase H-mediated Page 2 of 19 (page number not for citation purposes)