Thiols and disulfides in the tumor microenvironment: characterization of quiescin sulfhydryl oxidase 1 (QSOX1) in the extracellular matrix
Date
2022
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Publisher
University of Delaware
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related deaths in the U.S., despite only being the 11th most common cancer. Coupled with a 5-year survival rate of only ~7%, PDAC is one of the most lethal cancers in the world. The high mortality rates of PDAC can be partially attributed to the tumor microenvironment. Unlike most carcinomas, PDAC is characterized by a strong desmoplastic reaction, or a mechanical stiffening of the extracellular matrix (ECM) in response to chronic inflammation. This desmoplastic reaction is mediated by cancer-associated fibroblasts that deposit ECM proteins (collagens, laminins, fibronectin, etc.) and matrix-remodeling proteins (MMPs, LOX, etc.) in the tumor parenchyma. Within the past decade, the enzyme quiescin sulfhydryl oxidase 1 (QSOX1) has gained recognition as significant contributor to solid tumor pathogenesis, but its biological role remains unclear. ☐ QSOX1 is a disulfide bond generating catalyst that participates in oxidative protein folding in the mammalian cell. QSOX1 is also overexpressed in solid tumors, including pancreatic and breast cancer. Current studies show that inhibiting or knocking down QSOX1 reduces pancreatic cancer cell migration and invasion, alters ECM deposition and organization, and decreases overall tumor growth in mice. However, it is unclear which features of the tumor microenvironment modulate QSOX1 and cause its overexpression in cancer. Additionally, few studies have differentiated between intracellular QSOX1 and secreted QSOX1 when interpreting data. QSOX1 has two isoforms: an intracellular version and a shorter, secreted version. The functionality of secreted QSOX1 may be critically different than its intracellular counterpart, but this is fundamentally not understood. ☐ Finally, thiols and disulfides on the surfaces of cells and ECM are involved in many biological processes, including adhesion, migration, and local invasion into the surrounding tissue. Polystyrene sulfonate (PSS) was recently found to significantly increase cell surface thiol labeling for high resolution fluorescent microscopy. This method of labeling thiols may be used to determine the thiol/disulfide redox state for various tissue culture platforms, including live cell culture. However, the effect of PSS chain length and concentration on labeling efficacy and cell viability has not been fully assessed yet. ☐ This dissertation is divided into three main aims that uncover the role of secreted QSOX1 and thiols/disulfides in the extracellular matrix. ☐ In aim 1, we investigated how QSOX1 is secreted into the ECM by pancreatic cancer cells. Solid tumors are known to release bioactive cargo through a variety of pathways, including those involving extracellular vesicles and particles (EVPs). We identified subpopulations of EVPs containing QSOX1 and determined the proportion of QSOX1 that is secreted through these alternate biogenesis pathways. We then used conventional protein purification techniques, including ultracentrifugation, tangential flow filtration, and ultrafiltration to separate conditioned media produced by pancreatic cancer cells to determine which fractions (EVPs, aggregates/complexes, or free protein) contained QSOX1. Finally, we investigated ECM protein-QSOX1 interactions in vitro. Through this research, we hypothesized a novel mechanism for QSOX1 in which it forms mixed disulfide bonds with cysteine-rich ECM proteins, a process that may be dysregulated in cancer. This would implicate QSOX1 as a matrix remodeling enzyme. ☐ In aim 2, we explored potential regulators of QSOX1 expression in cancer. We simulated two major features of PDAC: hypoxia and stiffness. To induce hypoxia, we exposed pancreatic cancer cells to atmospheric (low O2) and chemical (CoCl2) hypoxia for up to 48 hours. We observed that QSOX1 expression varied over time but was non-responsive to hypoxia. To stimulate stiffness, we used polyacrylamide gels and altered crosslinking amount and gel v/v% to create soft and stiff surfaces. We discovered that QSOX1 levels decreased on softer surfaces compared to conventional tissue culture plastic. ☐ In aim 3, we quantified thiol labeling efficacy of different molecular weight PSS on cell surfaces and determined its cytotoxicity at those concentrations. Interestingly, PSS is still used to treat hyperkalemia under the brand name Kayexalate, despite its known harmful side effects (including intestinal necrosis). We found that very low concentrations of 1 MDa PSS can enhance extracellular thiol labeling, but cell cytotoxicity becomes noticeable and significant above 10 µg/ml. This project expands upon a previous dissertation establishing that PSS increases thiol labeling on cell surfaces that is amenable for high-resolution microscopy. ☐ In summary, the work in this dissertation includes a thorough analysis of QSOX1 secretion from pancreatic cancer cells, potential regulators of QSOX1 in the tumor microenvironment, and an examination of thiols and disulfides in the ECM.
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Keywords
Disulfides, ECM, Extracellular vesicle, QSOX, Quiescin sulfhydryl oxidase, Tumor microenvironment