The proposed project aims to elucidate the epigenome of cancer stem cells in breast tumours in an effort to shed some light into the pathophysiology of these cells and with the ultimate goal of developing therapeutic schemes that target them specifically.Accumulating evidence suggests that only a subset of cancer cells, the CSCs, have the ability of self-renewal and can support cancer progression, while the rest of the tumour cells have only limited proliferative capacity and do not significantly contribute to tumour growth. Consequently, successfull anti-cancer treatments should seek to specifically eradicate these cells and not target the bulk of the tumour. To this end, it is critical that we reveal and study the unique genetic and epigenetic characteristics of CSCs. Epigenetic mechanisms are believed to drive the differentiation of CSCs to less malignant cells. Thus, differences in their epigenome may be accountable for their different tumourigenic potential.In the first aim of the proposed project we will perform analysis of the epigenome (DNA methylation, post-translational histone modifications and small non-coding RNAs) of breast CSCs and non-CSCs by next-generation sequencing. The data generated from these experiments are expected to reveal the unique epigenetic signature of breast CSCs.This information will be used in the second aim of this project to develop and validate an in vitro cell culture system that will mimick the in vivo state of CSCs. This system will be used for screening drugs that specifically target epigenetic mechanisms in CSCs, as opposed to current epigenetic treatments for breast cancer that target the phenotypic characteristics of the bulk of the tumor.The proposed project is expected to contribute significantly in a new exciting field, that of cancer stem cells, and to help the applicant reintegrate in the European research community after training and working at top Institutes of USA and Canada for many years.