Our research program is focused on the cellular response to stresses, with an emphasis on low oxygen (hypoxia), oxidative stress and endoplasmic reticulum (ER) stress. Recent research in our laboratory has focused on proteins that traffic from the ER to the cell surface (CS) and into extracellular vesicles (EVs), through the secretory pathway, under these stress conditions. With respect to hypoxia, our interest has been on enzymes that function as cellular “oxygen sensors”; enzymes that modify other proteins that utilize oxygen as a substrate. We endeavor to utilize our knowledge and experience in cellular oxygen sensing to elucidate the mechanisms of how non-activated (or quiescent) fibroblasts and cancer-associated fibroblasts (CAFs) remodel the extracellular matrix (ECM) under different oxygen regimes. The research combines the fields of Biochemistry, Biotechnology, Cell Biology, Engineering and Medicine.
1. The Role of Oxygen in Tissue Remodeling
Fibroblasts are remarkable cells and are the “tissue remodelers” of the body, having roles in fibrosis, cancer, autoimmunity and wound healing. They are the most common cells of connective tissues but have poorly defined lineages as there are a limited number of fibroblast-specific markers. They play an important role in producing the proteins of the ECM and in building the structural framework (stroma) for animal tissues. More recently, fibroblasts have been recognized as a highly diverse cell population, with many different functions, that constantly respond and adapt to their environment. Activated fibroblasts (termed myofibroblasts) produce the major components of the ECM including collagen, elastin, fibronectin, laminin, glycosaminoglycans and reticular and elastic fibres. The nature (stiffness, density, elasticity and composition) of the ECM they produce influences cell proliferation, differentiation, migration and polarization, as well as in the morphogenesis of vital organs. This is important in tumour formation as the ECM associated with tumours determines their biological responses (metastasis, chemoresistance, etc.). Quiescent fibroblasts associated with the tumour microenvironment (TME) become activated CAFs, which display features similar to myofibroblasts (markers, increased ECM production, etc.). CAFs are defined as cells that have an elongated spindle morphology, negative nonmesenchymal (epithelial, endothelial, and leukocyte) biomarkers, positive mesenchymal biomarkers (such as vimentin (VIM), alpha-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and platelet-derived growth factor alpha (PDGF-α)) and lack genetic mutations. In the early stages of tumour formation, CAFs have certain tumour-suppressive functions, but as the tumour progresses, CAFs evolve into a tumour-promoting phenotype and perform tumour-promoting functions from multiple aspects. These include secreting a) factors that participate in the generation and maintenance of cancer cell stemness, b) factors that regulate the immune system, c) angiogenic factors for blood vessel formation, d) ECM remodeling factors to facilitate metastasis and e) cytokines/growth factors that promote chemoresistance. CAFs thus remodel the ECM and structure of the TME to create a “stromal niche”, promoting the migration and residency of other types of cancer cells.
A key feature of the TME is hypoxia. Normal tissue oxygen levels vary within and among organs but typically fall in a range of 3–9%; substantially less than the 21% present in the atmosphere. Within cells, this is even lower, ranging from 1.3 to 2.5%. The partial pressure of oxygen is below 5 mmHg (<1 % oxygen) in over 50% of locally advanced solid tumours, compared to venous blood which has a partial pressure of oxygen at 40-60 mmHg (5-8 % oxygen) and our atmosphere (21 % oxygen; 160 mmHg). This lack of oxygen within solid tumours leads to the upregulation of hypoxia-inducible factors (HIFs), transcription factors that induce the transcription of thousands of genes activated in low oxygen, including those involved in angiogenesis, metastasis, anaerobic metabolism (production of glucose), increased cell migration and ECM remodeling. Known as the Warburg Effect, cancer cells with an elevated glucose metabolism produce lactate, which decreases the pH in the TME from a neutral and healthy 7.35-7.45 to an acidic 6.3-7.0. Very little is known of the effects of pH in ECM remodeling. In cancer, ECM remodeling leads to changes in tumour composition, density, and mechanical properties, affecting its aggressiveness and response to therapy.
Our laboratory investigates the oxygen-dependent modification of ECM components and how this affects the “stiffness” of the matrix itself. We investigate the extent of the production of the ECM by myofibroblasts and CAFs, under different oxygen regimes, and how this affects the biomechanical properties of the matrix itself. Projects involve the use of human lung fibroblasts and CAFs within different microenvironments that simulate normal and disease (lung cancer, asthma, viral infection, etc.) states.
2. Redox Control of Protein Structure and Function
Oxidative modification of proteins by reactive oxygen species (ROS) may either activate or inactivate various proteins that are involved in responses to high (hyperoxia) or low (hypoxia) oxygen. Others may be resistant to such nonspecific modifications, and thus retain function under physiological changes in cellular ROS content. Modifying ROS include superoxide, hydrogen peroxide, hydroxyl radical, nitric oxide and peroxynitrite. Projects involve cloning genes of target proteins, expression of proteins in mammalian cell lines, immunopurification of target proteins and protein complexes, and structure/function studies (which includes site-directed mutagenesis of key amino acids targeted for modification) of partially purified or purified proteins.