A new role for inflammatory peroxidases in breast cancer development and metastasis

Abstract

Breast cancer is the leading cause of cancer mortality in women worldwide. Although we have made significant improvements in the detection and treatment of localized breast cancer with women surviving longer than ever before, patients with metastatic disease have a far worse prognosis. Metastasis is the leading cause of death in breast cancer patients with a 5-year survival of less than 30% and is currently without a cure. Thus, improvements in the development of new treatments will have profound impact on outcomes for these patients. Recent insight into the interactions of stromal cells and cancer cells within the breast tumour microenvironment, such as immune cells, fibroblasts, extracellular matrix (ECM) proteins, and vasculature, have revealed that the stroma plays a critical role in regulating a favourable environment for breast cancer initiation, progression and metastasis. Therefore, aspects of stromal biology are the key focus for future studies to improve patient outcome, by identifying new targets for therapy development. The role of the innate immune infiltrates in regulating cancer progression has been receiving considerable attention over recent years. Clinical studies indicate the extent of neutrophil and eosinophil accumulation within the developing tumour microenvironment is strongly correlated with a poor patient prognosis and survival. Having infiltrated the tumour, these inflammatory cells deposit myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in abundance. These enzymes have mainly been studied in the context of providing oxidative defence against invading pathogenic microorganisms, and to-date the functional consequence of their heightened presence within the tumour microenvironment has long been unclear. The work in this thesis aims to provide a mechanistic link between inflammatory peroxidase enzymes MPO and EPO, influencing stromal cells within the tumour microenvironment leading to tumour development and metastasis. Using physiological relevant ranges of peroxidases this study revealed that peroxidases possess a well-conserved pro-fibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM using both in vitro and in vivo approaches. Structurally, the ECM generated upon peroxidase stimulation demonstrated highly linearized collagen fibers synonymous with tumour ECM that is highly conducive for cancer cell adhesion, and invasion. This suggests that peroxidase-mediated collagen biosynthesis by cancer-associated fibroblasts may play an important role in tumour progression. The stimulation of angiogenesis is considered to be one of the most important hallmarks of tumour progression. Data in this thesis demonstrates for the first time the proangiogenic capacity of both MPO and EPO to significantly enhance the in vitro proliferation, migration and capillary formation of human endothelial cells that are fundamental in the process of angiogenesis. The use of a murine model of angiogenesis confirmed the ability of these peroxidase enzymes to facilitate the generation of functional blood vessels in an in vivo model. These results suggest that MPO and EPO play a crucial role as drivers of angiogenesis, with the potential to support the vascularization of tumours and promote growth and metastasis. In addition, the pro-angiogenic and pro-fibrotic activity displayed by peroxidases was blocked using the peroxidase inhibitor 4-ABAH, indicating that the catalytic activity is essential for its activity. Using the orthotopic 4T1 mouse mammary carcinoma model, we demonstrated for the first time that delivery of MPO and EPO directly into developing tumours increased primary tumour burden and concomitant with enhanced lung metastases. Histological inspection confirmed that MPO and EPO stimulated an increase in endothelial cell recruitment, fibroblast activation and collagen deposition within these tumours. In conclusion, our findings demonstrate for the first time that the peroxidase enzymes MPO and EPO confer a broader range of action than previously thought and exhibit potent effects in the tumour milieu on matrix function, composition, angiogenesis, tumour invasion and metastasis. Importantly, these studies identify the use of peroxidase inhibitors that block the catalytic activity of the enzyme, as a potential novel therapeutic strategy for breast cancer prevention and therapy.