Changes in glycosylation have long been appreciated to be part of the cancer phenotype; sialylated glycans are found at elevated levels on many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to cell surface sialylation are not well characterized, specifically in bona fide human cancer. Metabolic and bioorthogonal labeling methods have previously enabled enrichment and identification of sialoglycoproteins from cultured cells and model organisms. The goal of this work was to develop technologies that can be used for detecting changes in glycoproteins in clinical models of human cancer.
In Chapter 1 of this dissertation, I present an overview of the structures and functions of glycans and their relationship to cancer progression. I also discuss applications of in vivo bioorthogonal labeling in model organisms and how in humans, the significant regulatory and ethical barriers associated with introducing chemically altered sugars into people have hindered it. Finally, I review mass spectrometry-based proteomics and how it can be applied to clinical glycoproteomics.
In Chapter 2, I demonstrate the first application of this bioorthogonal labeling in a glycoproteomics platform applied to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of functionalized derivatives of N-acetyl mannosamine, the sialic acid biosynthetic precursor. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of human clinical relevance.
Secretome proteins play important roles in regulation of many physiological processes and show utility as potential biomarkers and for noninvasive diagnostics and treatment monitoring. In Chapter 3, I discuss a platform for identifying sialoglycoproteins that were secreted in the conditioned media from bioorthogonally labeled human prostate tissue slice cultures. This platform could be used to identify disease biomarkers in a faithful clinical model of human disease.
Mutations in granulocyte colony-stimulating factor 3 receptor (CSF3R), also known as G-CSFR, occur in the majority of patients with chronic neutrophilic leukemia (CNL) and are more rarely present in other kinds of leukemia. In Chapter 4, I discuss novel variants in CSF3R at asparagine residue N610, one of which was germline. Interestingly, these N610 substitutions are potently oncogenic and result in ligand-independent receptor activation. They confer activation of the JAK-STAT signaling pathway and concurrent sensitivity to JAK kinase inhibitors. The N610 residue is part of a consensus N-linked glycosylation motif in the receptor. Detailed mass spectrometry analysis demonstrates that this site is occupied by both complex and complex bisecting glycans. Further analysis demonstrates that N610 is the primary site of sialylation of the receptor. This study demonstrates that membrane-proximal N-linked glycosylation is critical for maintaining the ligand dependence of the receptor. Furthermore, it expands the repertoire of potently oncogenic mutations in CSF3R that are therapeutically targetable.
|Advisor:||Bertozzi, Carolyn R., Wemmer, David E.|
|Commitee:||Francis, Matthew B., Herr, Amy E.|
|School:||University of California, Berkeley|
|School Location:||United States -- California|
|Source:||DAI-B 79/08(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Oncology|
|Keywords:||Bioorthogonal chemistry, Glycoproteomics, Glycosylation, Metabolic incorporation, Prostate cancer, Proteomics|
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