Download Ph.D. Student: Anne Geske Lindhard Christensen Enrolment: 1

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