To explore the mechanisms whereby estrogen and antiestrogen (tamoxifen (TAM)) can regulate breast cancer cell growth, we investigated gene expression changes in MCF7 cells treated with 17 -estradiol (E 2 ) and/or with 4-OH-TAM. The patterns of differential expression were determined by the ValiGen Gene IDentification (VGID) process, a subtractive hybridization approach combined with microarray validation screening. Their possible biologic consequences were evaluated by integrative data analysis. Over 1000 cDNA inserts were isolated and subsequently cloned, sequenced and analyzed against nucleotide and protein databases (NT/NR/EST) with BLAST software. We revealed that E 2 induced differential expression of 279 known and 28 unknown sequences, whereas TAM affected the expression of 286 known and 14 unknown sequences. Integrative data analysis singled out a set of 32 differentially expressed genes apparently involved in broad cellular mechanisms. The presence of E 2 modulated the expression patterns of 23 genes involved in anchors and junction remodeling; extracellular matrix (ECM) degradation; cell cycle progression, including G 1 /S check point and S-phase regulation; and synthesis of genotoxic metabolites. In tumor cells, these four mechanisms are associated with the acquisition of a motile and invasive phenotype. TAM partly reversed the E 2 -induced differential expression patterns and consequently restored most of the biologic functions deregulated by E 2 , except the mechanisms associated with cell cycle progression. Furthermore, we found that TAM affects the expression of nine additional genes associated with cytoskeletal remodeling, DNA repair, active estrogen receptor formation and growth factor synthesis, and mitogenic pathways. These modulatory effects of E 2 and TAM upon the gene expression patterns identified here could explain some of the mechanisms associated with the acquisition of a more aggressive phenotype by breast cancer cells, such as E 2 -independent growth and TAM resistance.

To understand the cellular mechanisms of malignant transformation induced by constitutive activation of the ras oncogene (Ha-ras), we used a subtractive hybridization method (VGID TM ) together with an integrative analytical procedure based upon literature databases in the form of extensive interaction graphs. We found 166 over-and underexpressed genes which, in the human MCF7-ras breast epithelial cell line, are involved in the different aspects of tumoral transformation such as de®ned signaling pathways, cellular growth, protection against apoptosis, extracellular matrix and cytoskeleton remodeling. Integrative analysis led to the construction of a physiological model de®ning cross-talk and signaling pathway alterations which explicitly suggested mechanisms directly involved in tumor progression. The model further suggested points and means of intervention which could induce cell death in Ha-ras-transformed cells speci®cally. These hypotheses were directly tested in vitro and found to be largely correct, hence indicating that these new analytical and technological approaches allow the discovery of pathology-associated cellular mechanisms and physiologically de®ned targets leading to phenotype-speci®c pharmacological interventions.