Proteomics and N-glycome analyses reveal insights into the pathogenesis of the molecular subtypes of breast cancer

Breast cancer is the most common type of cancer affecting women worldwide. It is the leading cause of cancer-related death among females and its incidence rate is rising sharply. Significant molecular heterogeneity exists within breast cancer, which consequently leads to the formation of multiple molecular subtypes of the disease. In an effort to address the challenges associated with establishing reliable markers predictive of breast cancer and to develop effective drug therapies, the major aim of this thesis is to achieve an improved understanding of the molecular mechanisms and pathway deregulation in the breast cancer pathology. The studies described in this thesis applied high throughput proteomics and glycomics analyses, which allowed parallel global protein and N-glycan comparisons, respectively, to be made to define discriminatory patterns that correlated with the molecular heterogeneity observed in breast cancer. Specifically, comparative proteomics and glycomics of secreted and membrane fractions from a panel of breast cancer cell lines corresponding to three common breast cancer subtypes including luminal A, HER2-enriched and basal B subtypes, were performed using nontumorigenic human mammary epithelial cells (HMEC) as a normal healthy reference. The distinctive subcellular proteome and glycome signatures unique to the individual cancer subtypes were functionally evaluated by utilizing a range of bioinformatics-assisted pathway analysis tools to gain insights into regulatory mechanisms underlying the normal and tumorigenic cellular processes. The combination of structural and functional proteomics yielded consistent molecular themes involved in the pathogenesis of breast cancer. In addition, distinctive molecular features associated with each subtypes were present. In the first study of its kind, comprehensive analysis of the secreted N-glycome of a panel of breast epithelial cells investigated the involvement of protein N-glycosylation in breast cancer. The causative and/or effector roles of aberrant N-glycosylation in breast tumorigenesis were evident as strongly supported by the presence of tumor-promoting N-glycan determinants in the secreted and membrane fractions of breast cancer cells. Significantly, unique secretome N-glycosylation signatures enabled breast cancer subtype classification. Subcellular-specific N-glycosylation was found to be a universal cellular feature not only limited to epithelial breast cancer cells and was mechanistically explained by the differential solvent accessibility to the asparagines residues forming the N-glycosylation sites. Having mapped this relationship between spatial accessibility and N-glycan processing of glycoproteins is important to allow us to understand the expression and (de)regulation of glycoepitopes in breast cancer. In recognizing the importance of investigating intact glycopeptides to integrate the information from the obtained breast cancer cellular proteome and glycome and obtain site-specific information of protein N-glycosylation of breast cancer cells in future work, a multi-lectin affinity chromatography platform for cancer-specific glycoprotein enrichment directly from whole cell 20lysates was developed and optimized, which will serve as a useful tool in glycoproteomics. In conclusion, this thesis provides the most detailed picture of the proteome and N-glycomede regulation in multiple breast cancer subtypes to date, which yields valuable insight into the multiple mechanisms associated with the pathophysiological changes in breast cancer. This molecular insight forms an important knowledge platform from which the emerging field of glycoproteomics promise to yield an even higher definition of the tumor-specific protein modifications and, as a consequence, eventually allow us to develop targeted molecular therapeutics and diagnostics tools to benefit the growing number of women affected by the disease.