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Ph.D. Student: Anne Geske Lindhard Christensen Enrolment: 1 February 2010 Project Title: Preclinical evaluation of proteomic biomarkers of tumor initiating cells and other hierarchical clusters of triple-negative breast cancer. Supervisors: Professor Henrik Ditzel, dr.med., ph.d.*, Associate Professor Rikke Leth-Larsen, ph.d*, Associate Professor Martin R Larsen, ph.d** *Institute of Molecular Medicine, SDU, **Institute of Biochemistry and Molecular Biology, SDU. Institute: Institute of Molecular Medicine Research Unit: Department of Cancer and Inflammation Mass spectrometry based investigation of the phosphoproteome of breast cancer stem cells. Protein kinases are among the most important oncogenes yet known. Following ligand binding, a series of phosphorylation cascades then serve to propagate the signal from the cell surface into the nucleus in a highly dynamic process. The phosphorylation of proteins play key roles in the regulation of cancer cells, and protein kinases are therefore targets of several new cancer drugs and drug candidates. A characterization of the phosphorylation pattern of signaling pathways in cancer cells and specially the breast cancer stem cells are therefore needed. This study is based on a cell line model consisting of a panel of isogenic cell lines made from single cell cloning of a triple-negative breast carcinoma cell line. The model includes cell lines with cancer stem cell characteristics as they are able to generate primary tumors when inoculated in the mammary fat pad of immune deficient mice, give rise to more differentiated cells of the tumor and have the ability to self-renew in mammospheres in vitro. In addition, the model includes a panel of cell lines which do not possess these properties, thus is considered to belong to the more differentiated bulk of the tumor. A cell line with cancer stem cell characteristics and a cell line without such characteristics were each fractionated in a membrane and a soluble fraction, the proteins purified and digested, and peptides from the two cell lines were dimethyl-labeled and mixed in a one to one ratio. Phosphoproteins were isolated from the fractionated mixtures using titanium dioxide (TiO2) and analyzed by mass spectrometry. We obtained a total of 3977 unique proteins of which 1239 were regulated (>1.5) fold between the two cell lines.2144 phosphoproteins were detected and of these were 1578 regulated. A comparison of the proteome and phosphoproteome analysis further established that at least 332 of these phosphoproteins were regulated only at the post translational level. In addition, overall 125 kinases and 58 phosphatases were identified, of these a significant amount was found to be regulated between the two cell lines. These data constitute the foundation of network analyses investigating regulated pathways between cancer stem cells and cells of the tumor bulk. Pathways of which some are potential targets for therapeutic strategies. Keywords: Oncology and Haematology