Gangliosides are sialic acid-containing glycosphingolipids expressed on all vertebrate cells. They are primarily positioned in the plasma membrane, with the ceramide part anchored in the membrane and the glycan part exposed on the surface of the cell. These lipids have highly diverse structures, in particular with respect to their carbohydrate chains, with N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) being the two most common sialic acid residues in mammalian cells. Generally, human healthy tissue is considered nearly deficient in NeuGc, but since this molecule is present in tumors and in human fetal tissues, it was earlier classified as an onco-fetal antigen. Gangliosides perform important functions through carbohydrate-specific interactions with proteins, for example as receptors in cell-cell recognition, which can be exploited by viruses and other pathogens, and also by regulating signaling proteins through lateral interaction in the membrane. Through both mechanisms, tumor-associated gangliosides may affect malignant progression, which makes them attractive targets for cancer
In this thesis, the ganglioside NeuGc GM3 is the focus of attention. Different approaches have been used in order to reveal both its importance in cancer progression but also its potential for cancer immunotherapy. In particular, we investigated the effect of hypoxia on tumor cells, since hypoxic conditions are known to induce a change in carbohydrate determinants correlating with a more aggressive and therapy-resistant phenotype. We studied alterations in protein expression as a response to hypoxia, using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS/MS. The results obtained from the quantitative proteome analysis uncovered proteins important in hypoxia-induced cancer progression, representing potentially interesting targets in future therapies. The same method (SILAC) was used in order to study the cell death killing mechanism employed by a promising monoclonal antibody (14F7) that specifically targets NeuGc GM3. The identified proteins advance our understanding of this novel mechanism of cell killing. In addition, we studied the molecular interaction between the antibody 14F7 and its antigen NeuGc GM3 or its anti-idiotypic antibody using a combination of mass spectrometry-based methods and surface plasmon resonance (SPR).
List of papers I-IV. The papers are removed from the thesis due to publisher restrictions.
I. Paula A. Bousquet, Joe A. Sandvik, Magnus Ø. Arntzen, Nina F. Jeppesen Edin, Stine Christoffersen, Ute Krengel, Erik O. Pettersen, Bernd Thiede. Hypoxia strongly affects glycolysis and expression of mitochondrial proteins, as shown by quantitative proteomics of HeLa cells. In Manuscript.
II. Paula A. Bousquet, Joe A. Sandvik, Nina F. Jeppesen Edin, Ute Krengel. How does hypoxia trigger the expression of NeuGc GM3? In Manuscript.
III. Paula A. Bousquet, Joe A. Sandvik, Magnus Ø. Arntzen, Bernd Thiede. Ernesto Moreno, Ute Krengel. SILAC analysis of HeLa cells treated with the anti-tumor monoclonal antibody, 14F7. In Manuscript.
IV. Paula A. Bousquet, Nebiyu Abshiru, Matthiew Tessier, Rune F. Johansen, André van Eerde, Karin Lindkvist, Magnar Bjørås, Bernd Thiede, Robert J. Woods, Ernesto Moreno, Ute Krengel. MS and SPR analysis of the molecular interaction of a unique anti-tumor antibody with its antigen N-glycolyl GM3 and an anti-idiotypic antibody. In Manuscript.