title: Annexin-A1 signalling in inflammation and cancer
Creators: name: Kelly, Lauren
Publisher: name: University of Glasgow
Year: 2022

description: Annexin-A1(ANXA1) is a member of the Annexin family of calcium-dependent phospholipid-binding proteins that functions in a range of biological processes from cell proliferation to apoptosis. This glucocorticoid-induced protein has a well-defined anti-inflammatory role in innate immunity, with unclear functions in adaptive immunity due to the limited, contrasting data available. A role for ANXA1 and the formyl peptide receptors (FPRs) through which it signals has been suggested in a range of inflammatory diseases and cancers. This thesis aimed to investigate the ANXA1-FPR signalling axis in two primary examples of these disease settings in which pathogenesis is far from understood: Psoriatic Arthritis (PsA) and Multiple Myeloma (MM). Moreover, this project aimed to identify the impact of addition of a novel anti-ANXA1 antibody, MDX-124, to the immune response in healthy cells. Initial work revealed that MDX-124 could bind to a range of immune cell subsets and confirmed high binding of MDX-124 to cells known to express high amounts of ANXA1 (monocytes). Functional analysis revealed that addition of MDX-124 to both stimulated THP-1 monocytic cells and primary monocytes was associated with an increase in pro-inflammatory cytokine production. Moreover, addition of MDX-124 during the differentiation of THP-1s resulted in an increased activation of intracellular and extracellular signalling proteins associated with survival. This provided some insight into the potential mechanism of how MDX-124 could function in inflammatory settings. To investigate the potential role for ANXA1-FPR signalling in PsA, preliminary studies characterised surface expression levels of ANXA1 and its receptors, FPR1 and FPR2 on a range of immune cell subsets in PsA and healthy control (HC) samples. This analysis confirmed altered expression of ANXA1, FPR1 and FPR2 in disease-relevant cells PsA. Moreover, analysis of RNA sequencing data form PsA lesional skin (PsA L) revealed an increase in FPR1 and FPR2 in these samples compared to HCs. Further analysis defined key disease-associated pathways that were enriched in the PsA L skin associated with FPR1 and FPR2 signalling. This analysis implicated involvement of the ANXA1-FPR signalling axis in both the skin and peripheral blood compartments in PsA, however more samples are required to confirm this preliminary analysis. To investigate the role of ANXA1-FPR signalling in MM, flow cytometric analysis of ANXA1, FPR1 and FPR2 surface expression in the immune cell compartment of of MM patient samples and HCs were assessed. Preliminary results revealed a substantial upregulation of both ANXA1 and FPR1 on the circulating B cells of MM patients in 1/3 MM samples assessed. This data was; therefore, inconclusive and further samples are required to investigate these differences expression levels of ANXA1 and FPR1 in MM circulating B cells. Analysis of genomic data from a publicly available MM database revealed that higher expression of the ANXA1 gene at baseline was associated with worst survival outcomes in the cohort of patients assessed. Moreover, high ANXA1 expression was associated with an upregulation of genes associated with worst prognosis and treatment resistance in MM. Notably, baseline ANXA1 expression correlated with the 1q21 chromosomal amplification abnormality in this cohort of patients, which is linked to worst prognosis in MM. Additionally, high ANXA1 was associated with an upregulation of the S100 family of calcium binding proteins, located at the 1q21 chromosome locus, which, in turn, have been associated with treatment resistance and worse prognosis in MM. This preliminary data has highlighted the potential of ANXA1 signalling in treatment response pathways in MM and provides rationale for further investigation into the interactions between ANXA1 and the MM- associated upregulated genes identified in this study. In summation, this preliminary data has identified key expression differences in ANXA1 and its receptors in both PsA and MM and highlighted potential pathways worth focusing on for future analysis. Additionally, more repeats of the experiments carried out in this study are needed to confirm the significance of these findings.