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S100A8 is an anti-inflammatory oxidant scavenger protein that induces IL-10 in airway epithelial cells in normal lungs following inhalation. These mechanisms contribute to its ability to attenuate endotoxin-induced acute lung injury by suppressing proinflammatory mediator induction. Sustained chronic inflammation promotes activation of myeloid-derived suppressor cells (MDSC) to reduce T cell surveillance by producing toxic reactive oxygen species and nitric oxide, and depleting the essential nutrient, arginine. MDSC accumulation facilitates tumour progression and is associated with reduced survival in lung cancer. S100A8 is upregulated in human lung cancer and the associations of S100 proteins with MDSC suggest a potential immunomodulatory role. We proposed that the anti-inflammatory and antioxidative properties of S100A8 improved outcomes in lung cancer. Remarkably, repeated S100A8 inhalation prolonged survival by up to 40% in mice implanted with orthotopic lung cancer (from 19±1 to 27±1 days). Tumour-bearing mice treated with S100A8 harvested at earlier time points were compared with vehicle-treated controls to identify potential changes in the microenvironment. At mid-point of survival, S100A8 significantly suppressed key cytokines that promote MDSC expansion and activation, with concomitant reduced MDSC numbers in lungs and spleen. Importantly, S100A8 also increased CD4 and NK-T cell numbers in these organs, suppressed nitrite levels and selectively enhanced activities of key antioxidant enzymes in lungs. S100A8 also upregulated thioredoxin reductase activity in lungs 3 days post-inhalation. Results suggest that S100A8 created a favourable microenvironment for effector T cell recruitment and function. Lung cancer is generally associated with poor prognosis and with limited treatment options, and S100A8 may enhance immunoprotection. Its delivery in combination with currently-available treatments may improve clinical outcomes.

Malignant transformation in squamous cell carcinomas (SCC) such as those of the head and neck (HNSCC) remains an enigmatic process that results in abnormal cellular differentiation, loss of growth and cell cycle regulation, gain of replicative immortality and resistance to apoptosis, activation of cellular migration and invasion, increase in energy metabolism, and evasion of immune destruction. These abnormalities in cell functions emerged as the hallmarks of cancer. Calprotectin, a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex (EDC), may play essential roles in the regulation of cell differentiation, cell cycle progression, cellular survival and cell migration that are part of the cancer hallmarks. While highly upregulated in a variety of cancers, calprotectin is down-regulated in squamous cell carcinomas of the cervix, esophagus and the head and neck. Using microarray analysis for gene expression we found that the S100A9 subunit of calprotectin was significantly down-regulated along with other EDC genes in human primary HNSCC cases, suggesting a loss of functional S100A8/A9 protein complex and differentiation during carcinogenesis. Expression of S100A9 correlated strongly with a set of HNSCC downregulated genes putatively involved in loss of cytodifferentiation and control of cell cycle. To probe its role in carcinogenesis, S100A8/A9 was stably expressed in a calprotectin-negative human carcinoma cell line (KB cells). Expression of S100A8/A9 in KB cells up-regulated differentiation and cell-cell contact growth inhibition signaling pathways, re-established epithelial actin microfilament cytoskeletal structures and v cellular adhesion to the extracellular matrix, down-regulated anti-apoptosis gene networks, and suppressed anchorage-independent survival and stress-induced (by serum starvation) cell migration. Calprotectin appeared to induce growth suppression by signaling a reactivation of G2/M cell cycle checkpoint regulators, Chk1 and PP2A, and inactivation of mitotic activators Cdc25C and Cdc2. As a result, we saw a marked increase in Cdc2-PThr14/Tyr15 phosphorylation and down-regulation of cyclin B1, suggesting an inactivation of the mitotic entry promoting Cdc2/cyclin B1 complex, resulting in cell cycle and mitotic arrest at the G2/M checkpoint. When inoculated into nude mice, KB cells producing calprotectin showed reduced tumor growth when compared to sham-transfected control KB cells. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increased carcinoma growth and survival and reduced Cdc2 phosphorylation at Thr14/Tyr15. Calprotectin expression in KB and TR146 cells also down-regulated expression of putative HNSCC marker genes, INHBA, PTGS2 (Cox-2) and SULF1, found to be expressed only in HNSCC samples. Calprotectin-mediated control of cellular differentiation and G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may point to new molecular targets for therapy.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

Mast cells (MCs) play a central role in inflammation by releasing mediators following activation. S100A8 and S100A9 are abundantly expressed in inflammatory sites such as asthmatic lung, sunburnt skin and atherosclerosis where MCs are involved in pathogenesis; roles of S100A8 in MC function are undetermined. The aims of this thesis were to determine effects of S100A8 on MC activation, particularly provoked by IgE and UVB. Initially, effects of UVB on MC activation were investigated as detailed functions were unclear. Cord blood-derived human mast cells (CBMCs) were treated in vitro with varying doses of UVB and production of multiple cytokines and viability investigated. UVB exposure selectively increased levels of IL-8 (CXCL8), and to a less extent IL-1β, but not eight other cytokines tested. New protein synthesis partially contributed and IL-8 production was p38 MAPK-dependent. UVB dose-dependently induced MC apoptosis indicating a potential regulatory mechanism of MC function. The ability of recombinant S100A8, S100A9 or S100A8/9 heterodimer to modulate IgE/antigen (DNP/anti-DNP)-mediated activation of a murine MC line, and of bone marrow-derived (mBM) MC activation was determined. The S100s did not directly induce degranulation or induce IL-6. S100A8 significantly inhibited DNP/anti-DNP-provoked degranulation, and IL-6 and TNF mRNA and protein induction. S100A8 did not alter FcεRIα expression. S100A9 was less effective; and the S100A8/9 complex was also suppressive. S100A8 only weakly suppressed non-specific MC degranulation. Mutation of Cys41 in S100A8 negated its suppressive activity. Because S100A8 scavenges oxidants via this reactive Cys residue, we propose that this may mediate its ability to downmodulate IgE-dependent MC responses. Similar to the thiol scavenger N-acetyl-L-cysteine, S100A8 but not the Ala41 mutant, attenuated DNP/anti-DNP-provoked LAT phosphorylation. However, the disulfide-bonded S100A8 dimer and S100A8 containing a sulfinamide bond between Cys41 and Lys34/35 also reduced MC activation, indicating an additional pathway(s). S100A8 did not suppress antigen/IgE-induced responses of CBMC possibly because these may not truly reflect fullymature human tissue MCs. S100A8 did not alter UVB-induced IL-8 release by CBMCs, or affect apoptosis. Murine S100A8 may have anti-inflammatory properties by regulating MC activation in an activator-specific manner, at least partially by scavenging ROS to suppress intracellular signalling.