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oai:keep.lib.asu.edu:node-1982002024-12-23T18:01:48Zoai_pmh:alloai_pmh:repo_items

198200 https://hdl.handle.net/2286/R.2.N.198200 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2024 165 pages Doctoral Dissertation Academic theses Text eng Koelbel, Calvin Lake, Douglas Borges, Chad Chen, Qiang "Shawn" Ho, Thai Arizona State University Partial requirement for: Ph.D., Arizona State University, 2024 Field of study: Molecular and Cellular Biology Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes disulfide bond formation in protein folding. Over the past decade, QSOX1 has emerged in the literature as a contributor to cancer invasion as well as a potential biomarker for several cancer types and acute decompensated heart failure (ADHF). The goals of this research were to develop tools for evaluating the potential for QSOX1 to serve as a biomarker for cancer progression and to investigate the role that QSOX1 plays in invasion with focus on differential function of QSOX1 isoforms. Tandem antigen capture enzyme-linked immunosorbent assays (ELISAs) measuring QSOX1 isoform levels in serum and plasma were generated using QSOX1 monoclonal antibodies (mAbs). One of these ELISAs was employed to measure QSOX1 levels in samples from patients with either bladder cancer or ADHF. Total QSOX1 alone was not a viable marker for bladder cancer progression or ADHF, but these ELISAs may still have clinical utility as additional analyses are implemented. Next, chemical and antibody inhibitors were pursued as methods to probe for the biological function of QSOX1. Analogs of a small molecule inhibitor of QSOX1 (SPX-9) and an inhibitory mAb were screened for suppression of the enzymatic activity of QSOX1 and cancer cell invasion. A more potent inhibitor of invasion was synthesized which was found to lack specificity to QSOX1 but could serve as a potent anti-cancer therapeutic. The inhibitory mAb suppressed invasion and tumor growth but has the limitation of inhibiting only extracellular QSOX1. To achieve complete silencing of QSOX1 and the ability to probe QSOX1 isoforms, CRISPR base editors were utilized to generate isoform-specific knockout (KO) cell lines by strategically introducing nonsense mutations and mutating splice sites. These QSOX1 isoform KO cells were assessed for cancer phenotypes and cellular pathways. Lastly, these KO cells were used to evaluate the ability of the commercially available and commonly used ProteinTech QSOX1 antibody to specifically detect QSOX1. Data suggest that the ProteinTech antibody may not detect QSOX1 nor be specific to QSOX1. This work has contributed novel tools and insights for understanding the diagnostic potential and cancer biology of QSOX1. Cellular Biology Oncology Molecular Biology Base Editing ELISA Invasion Isoforms QSOX1 Small Molecule Inhibitors Investigating the Diagnostic Potential and Cancer Biology of Quiescin Sulfhydryl Oxidase 1