Neutrophils and cancer: regulation of cell death and epigenetic pathways

Neutrophils constitute a significant proportion of recruited immune cells in many tumours. While activated neutrophils are able to exert robust anti-tumoural effects, more often they provide growth and survival signals that promote tumour progression. The mechanisms by which neutrophils impact tumours are unclear. The relationship between the immune system and cancer is particularly evident in cutaneous squamous cell carcinoma (CSCC). To investigate the role and potential prognostic value of neutrophils in CSCC, neutrophil numbers in circulation and in the tumours of non-therapeutically immuno-suppressed individuals was examined for associations with markers of increased risk of metastasis and overall survival. A circulating neutrophil count of greater than 3.5 x109 per L was associated with decreased overall survival. Moreover, three out of the four patients that developed metastases in the study cohort had circulating neutrophil counts above this threshold. When tumour tissue was examined for CD66b positive and myeloperoxidase (MPO) positive neutrophils, there was a significantly higher number of neutrophils in tumours with a thickness greater or equal to 5mm. Although there were no significant associations with the number of MPO positive cells or levels of extracellular MPO and high-risk tumour characteristics, MPO was present in all CSCC tumours, particularly in necrotic core regions. This suggests that neutrophils could have a role in the progression of CSCC. It also highlights that MPO from activated or dead neutrophils was present in the CSCC tumour microenvironment, and could have a functional role in promoting tumour development. Neutrophils can undergo necrotic cell death, which promotes tissue damage and an acute inflammatory response. There is considerable interest in neutrophil extracellular trap (NET) release in cancer, as there is mounting evidence to suggest that NETs can facilitate tumour metastasis. NET formation is a regulated event, and while some of the molecular mechanisms that lead to NET extrusion are known, others, such how the membrane becomes permeabilised, are still unclear. Necroptosis is a form of regulated necrosis that depends on mixed lineage kinase domain like pseudokinase (MLKL) forming an oligomeric complex at the plasma membrane. This study showed that TNFa induces necroptosis in human neutrophils. Pre-treatment of neutrophils with an MLKL inhibitor impeded TNFa mediated necroptosis and decreased NET release in phagocytic neutrophils, but not in PMA-stimulated neutrophils. These findings could not be corroborated by using the peripheral blood neutrophils of MLKL knockout mice, due to challenges encountered with their stimulation and assessment of cell death. Further research is required into the emerging roles of MLKL as well as the potential impact of oxidants on the molecular players of this pathway. Activated neutrophils produce hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) that can impact neighbouring cells and contribute to oxidative stress. Oxidative stress is considered a common feature of inflammation-driven cancers, and promotes genomic instability and aggressive tumour phenotypes. Altered DNA methylation can modify gene expression and cell function and can be influenced by environmental factors. DNA methyltransferases (DNMTs) and levels of the methyl donor, S-adenosyl methionine (SAM) can be regulated by oxidative stress. The effect of H2O2 and glycine chloramine (a product of the reaction of HOCl with glycine) exposure on short-term and heritable methylation patterns in proliferating T-lymphoma cells were investigated. Using bead chip array technology, differential methylation was assessed at 850,000 individual methylation sites (CpGs) across the human genome. Glycine chloramine and H2O2 exposure resulted in large genome-wide decreases in methylation after completion of replication, but most of these changes were corrected during subsequent rounds of cell division. However, many smaller methylation changes were conserved. The failure to restore correct methylation patterns could lead to the propagation of aberrant patterns and abnormal gene transcription. Notably, exposure to oxidants resulted in increased variability of methylation patterns in the cell population, indicating that not all methylation sites were affected in the same way. This finding may have important implications for cancer therapies, as genetic diversity is at the root of tumour heterogeneity, the evolution of drug-resistance, and poor clinical outcomes