Doctoral Dissertation Defense – Catherine S. Millar-Haskell

BME PhD Candidate Catherine S. Millar-Haskell will be defending her dissertation:


Thiols and Disulfides in the Tumor Microenvironment: Characterization of Secreted Quiescin Sulfhydryl Oxidase 1 (QSOX1) In the Extracellular Matrix



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, 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 role is fundamentally not understood.

This dissertation is divided into three main aims to 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 pathways. We then determined which fractions (EVPs, aggregates/complexes, or free protein) contained QSOX1. Finally, we determined with which ECM proteins QSOX1 interacts. Through this research, we hypothesized a novel mechanism for QSOX1 in which it forms mixed disulfide bonds with cysteine-rich ECM proteins, which may be dysregulated in cancer. This implicates 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. 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 is downregulated on softer surfaces compared to conventional tissue culture plastic. We also measured the enzymatic activity of secreted QSOX1 to show that secreted QSOX1 is functionally active.

In aim 3, we characterized the ability of polystyrene sulfonate (PSS) to enhance thiol labeling on cell and ECM surfaces and determined its cytotoxicity. We found that low concentrations of 1 MDa and 200 kDa molecular weight PSS can enhance extracellular thiol labeling, but cell cytotoxicity becomes noticeable and significant in the upper range of our working concentrations. This study expands upon previous work establishing PSS as a novel agent to enhance fluorescent labeling of thiols in the ECM for high-resolution microscopy.