This project aims at unraveling genetic and epigenetic events occurring along early stages of tumor transformation. It is known that genome of tumor cells undergo severe changes at the epigenetic (global demethylation of the genome and focused methylation of certain promoters) and structural (copy number and chromosomal integrity) levels. These epigenetic and genetic changes are believed to bear profound consequences at the mRNA and miRNA expression level. However, most data available stem from studies on human tumors or established cancer cell lines. Thus, little is known on the sequence of events accompanying the transition from normal to tumor cell. In this project we will study early steps of mammary epithelial cell cancer transformation. To this aim we plan to develop a series of in vitro models based on hMEC (human mammary epithelial cells) that we will progressively transform by sequential transduction of oncogenes and/or shRNAs. Cells will be obtained by primary culture of mammary explants obtained from patients who underwent plastic surgery. Transduced hMEC variants will be characterized at regular stages during the transformation process. We will establish their genetic (array-CGH and mRNA expression) profiles, whole genome DNA methylation (methylated DNA immunoprecipitation and oligo-arrays hybridization) and their miRNA expression profiles. We foresee to address the following questions: (1) are the different epigenetic and genetic events, occurring along the scheme of tumor transformation, coordinated' (2) are these changes modulated according to the oncogenic pathway initially activated to transform the cells' (3) identify novel cancer genes and cascades of genetic and epigenetic events that cooperate to lead to cancer transformation. We will establish 3 hMEC models transformed with combinations of shRNA and oncogenes that respectively result in the activation of distinct signaling pathways. Methylation, array-CGH, mRNA and miRNA profiles will be established and compared in order to determine whether differences exist among these hMEC variants. We will also analyse closely the sequences of events in order to determine whether epigenetic and genetic modifications occur stochastically or in a predetermined sequence. Finally, we will undertake functional studies to characterize candidate genes or genetic cascades identified in our screens.