Date of Award

2016

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

College of Arts and Sciences

First Advisor

Qian Wang

Abstract

Breast cancer is the second-most common cancer and the second-leading cause of cancer-related deaths in women. Despite advances in cancer early detection, prevention and treatment, breast cancer is still a major health challenge due to low survival caused by breast cancer metastasis. This warrants critical attention and intervention. From the proteomic standpoint, a protein-based multiplex system that provides large array of informative signals for cancer identification and prognosis is still limited. In this dissertation work, we developed two mass spectrometry-based strategies involving chemical biology tools for rapid protein fingerprinting of breast cancer cell lines, and for probing the O-linked N-acetylglucosamine (O-GlcNAc) proteome in transforming growth factor-beta (TGF-ß) induced epithelial-mesenchymal transition (EMT), a process that initiates metastasis. Investigation of O-GlcNAc EMT proteomics is critical in understanding how aberrant O-GlcNAc post-translational modification (PTM) promotes cancer invasion and metastasis, as well as in the identification of early stage therapeutic targets. Until now the role of O-GlcNAc PTM in TGF-ß-induced EMT is unknown.

In Chapter 2, a novel ‘one-step cell processing’ method was developed as a prerequisite to rapid spectral profiling of mammalian cells using Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). Upon analysis of the mass spectral data of breast cancer cell lines with pattern recognition methods, discrimination between metastatic and non-metastatic cell lines was accomplished, demonstrating the potential of MALDI-MS profiling in breast cancer diagnosis.

Chapter 3 reports a cleavable azide-reactive dibenzocyclooctyne-disulphide agarose-based beaded resin in Copper-free Click chemistry-based affinity enrichment of O-GlcNAc proteome from azido-GlcNAc labeled cellular extracts, that enabled the global O-GlcNAc proteomic profiling by shortgun proteomics with liquid chromatography-tandem mass spectrometry identification and label-free quantification. From TGF-ß-induced EMT in MNuMG cells 196 proteins were identified. 125 of these were putative O-GlcNAc proteins, 75% of which have been previously identified among O-GlcNAc affinity enrichment samples. Downstream bioinformatics analyses of the O-GlcNAc proteome data were performed using Ingenuity Pathway Analysis (IPA) software. In silico protein-protein interactions revealed a regulatory network for metastasis, while the most significantly represented metabolic and signaling pathways included glycolysis and several TGF-ß non-canonical pathways, respectively. A metastatic regulatory network that features core regulators β-catenin and cyclin-D1 both of which are regulated by O-GlcNAc transferase supports published study that shows that “O-GlcNAcylation Plays Essential Role in Breast Cancer Metastasis,” has led us to hypothesize that TGF-ß signaling cooperates with O-GlcNAc signaling in promoting EMT, invasion and metastasis, pending O-GlcNAc site-mapping and validation of the proteomic data.

Rights

© 2016, Matsepo Ramaboli

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