S100A8 and S100A9 in Cancer

S100A8 and S100A9 are Ca²⁺ binding proteins that belong to the S100 family. Primarily expressed in neutrophils and monocytes, S100A8 and S100A9 play critical roles in modulating various inflammatory responses and inflammation-associated diseases. Forming a common heterodimer structure S100A8/A9, S100A8 and S100A9 are widely reported to participate in multiple signaling pathways in tumor cells. Meanwhile, S100A8/A9, S100A8, and S100A9, mainly as promoters, contribute to tumor development, growth and metastasis by interfering with tumor metabolism and the microenvironment. In recent years, the potential of S100A8/A9, S100A9, and S100A8 as tumor diagnostic or prognostic biomarkers has also been demonstrated. In addition, an increasing number of potential therapies targeting S100A8/A9 and related signaling pathways have emerged. In this review, we will first expound on the characteristics of S100A8/A9, S100A9, and S100A8 in-depth, focus on their interactions with tumor cells and microenvironments, and then discuss their clinical applications as biomarkers and therapeutic targets. We also highlight current limitations and look into the future of S100A8/A9 targeted anti-cancer therapy.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

Expression and Function of S100A8/A9 (Calprotectin) in Human Typhoid Fever and the Murine Salmonella Model

Background

Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.

Methods and principal findings

S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9^-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9^-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.

Conclusion

S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.     

Calprotectin (S100A8/9) as serum biomarker for clinical response in proof-of-concept trials in axial and peripheral spondyloarthritis

Introduction

Biomarkers complementing clinical evaluations may help to reduce the length and size of proof-of-concept (PoC) trials aimed to obtain quick “go/no go” decisions in the clinical development of new treatments. We aimed to identify and validate serum biomarkers with a high sensitivity to change upon effective treatment in spondyloarthritis (SpA) PoC trials.

Methods

The candidate biomarkers high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), pentraxin-3 (PTX-3), alpha-2-macroglobulin (alpha-2-MG), matrix metalloproteinase-3 (MMP-3), calprotectin, and vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA) in healthy controls (n = 20) and SpA patients before and after 2 weeks of infliximab (n = 18) or placebo (n = 19) treatment in cohort 1. Clinical outcome was evaluated at week 12. Results were validated in ankylosing spondylitis (AS) with infliximab (cohort 2, n = 21) and peripheral SpA with etanercept (cohort 3, n = 20).

Results

Serum levels of calprotectin, hs-CRP, PTX-3, VEGF (all P < 0.001) and MMP-3 (P = 0.062), but not IL-6 and alpha-2-MG, were increased in SpA versus healthy controls. Treatment with infliximab, but not placebo, significantly decreased calprotectin (P < 0.001) and hs-CRP (P < 0.001) levels, with a similar trend for MMP-3 (P = 0.063). The standardized response mean (SRM), which reflects the ability to detect changes over time, was high for calprotectin (−1.26), good for hs-CRP (−0.96) and moderate for MMP-3 (−0.52). Calprotectin and hs-CRP, but not MMP-3, were good biomarkers for treatment response in axial and peripheral SpA as evaluated and confirmed in cohort 2 and 3 respectively.

Conclusions

Calprotectin and hs-CRP are good serum biomarkers with high sensitivity to change upon effective treatment at the group level in small-scale, short term PoC trials in SpA.     

Inflammation: a novel mechanism for the transport of extracellular nucleotide-induced arachidonic acid by S100A8/A9 for transcellular metabolism

Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.     

S100A8 and S100A9 in human arterial wall. Implications for atherogenesis

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.     

Human S100A9 Protein Is Stabilized by Inflammatory Stimuli via the Formation of Proteolytically-Resistant Homodimers

S100A8 and S100A9 are Ca²⁺-binding proteins that are associated with acute and chronic inflammation and cancer. They form predominantly heterodimers even if there are data supporting homodimer formation. We investigated the stability of the heterodimer in myeloid and S100A8/S100A9 over-expressing COS cells. In both cases, S100A8 and S100A9 proteins were not completely degraded even 48 hrs after blocking protein synthesis. In contrast, in single transfected cells, S100A8 protein was completely degraded after 24 h, while S100A9 was completely unstable. However, S100A9 protein expression was rescued upon S100A8 co-expression or inhibition of proteasomal activity. Furthermore, S100A9, but not S100A8, could be stabilized by LPS, IL-1β and TNFα treatment. Interestingly, stimulation of S100A9-transfected COS cells with proteasomal inhibitor or IL-1β lead to the formation of protease resistant S100A9 homodimers. In summary, our data indicated that S100A9 protein is extremely unstable but can be rescued upon co-expression with S100A8 protein or inflammatory stimuli, via proteolytically resistant homodimer formation. The formation of S100A9 homodimers by this mechanism may constitute an amplification step during an inflammatory reaction.     

Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity

S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes. Both proteins regulate migration of these cells via modulation of tubulin polymerization. Calcium binding induces formation of (S100A8/S100A9)2 tetramers. The functional relevance of these higher-order oligomers of S100 proteins, however, is not yet clear. To investigate the importance of higher-order oligomerization for S100 proteins, we created a set of mutations within S100A9 (N69A, E78A, N69A+E78A) destroying the high-affinity C-terminal calcium-binding site (EF-hand II). Mutations in EF-hand II did not interfere with formation of the S100A8/S100A9 heterodimer as demonstrated by yeast two-hybrid experiments and pull-down assays. In contrast, mass spectrometric analysis and density gradient centrifugation revealed that calcium-induced association of (S100A8/S100A9)2 tetramers was strictly dependent on a functional EF-hand II in S100A9. Failure of tetramer formation was associated with a lack of functional activity of S100A8/S100A9 complexes in promoting the formation of microtubules. Thus, our data demonstrate that calcium-dependent formation of (S100A8/S100A9)2 tetramers is an essential prerequisite for biological function. This is the first report showing a functional relevance of calcium-induced higher-order oligomerization in the S100 family.     

S100A8/A9 (Calprotectin) Negatively Regulates G2/M Cell Cycle Progression and Growth of Squamous Cell Carcinoma

Malignant transformation results in abnormal cell cycle regulation and uncontrolled growth in head and neck squamous cell carcinoma (HNSCC) and other cancers. S100A8/A9 (calprotectin) is a calcium-binding heterodimeric protein complex implicated in cell cycle regulation, but the specific mechanism and role in cell cycle control and carcinoma growth are not well understood. In HNSCC, S100A8/A9 is downregulated at both mRNA and protein levels. We now report that downregulation of S100A8/A9 correlates strongly with a loss of cell cycle control and increased growth of carcinoma cells. To show its role in carcinogenesis in an in vitro model, S100A8/A9 was stably expressed in an S100A8/A9-negative human carcinoma cell line (KB cells, HeLa-like). S100A8/A9 expression increases PP2A phosphatase activity and p-Chk1 (Ser345) phosphorylation, which appears to signal inhibitory phosphorylation of mitotic p-Cdc25C (Ser216) and p-Cdc2 (Thr14/Tyr15) to inactivate the G2/M Cdc2/cyclin B1 complex. Cyclin B1 expression then downregulates and the cell cycle arrests at the G2/M checkpoint, reducing cell division. As expected, S100A8/A9-expressing cells show both decreased anchorage-dependent and -independent growth and mitotic progression. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increases growth and survival and reduces Cdc2 inhibitory phosphorylation at Thr14/Tyr15. The level of S100A8/A9 endogenous expression correlates strongly with the reduced p-Cdc2 (Thr14/Tyr14) level in HNSCC cell lines, SCC-58, OSCC-3 and UMSCC-17B. S100A8/A9-mediated control of the G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may suggest new therapeutic approaches.     

Loop A Is Critical for the Functional Interaction of Two Beta vulgaris PIP Aquaporins

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.     

S100A8/S100A9 and their association with cartilage and bone

S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.     

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.     

A comparison of human S100A12 with MRP-14 (S100A9)

MRP-8 and -14 are two S100 proteins highly expressed as a complex by neutrophils, and to a lesser extent by monocytes and certain squamous epithelia. However, less is known about the close homologue S100A12. This S100 protein is expressed by neutrophils and here we show that it is also expressed by monocytes, but not lymphocytes. An absence of coimmunoprecipitation of MRP-14 and S100A12 indicates that S100A12 is not associated with the MRP proteins in vivo. When directly compared to MRP-14, S100A12 expression by squamous epithelia is more restricted. In esophagus and psoriatic skin, S100A12 is differentially regulated, like MRP-14, but the expression pattern of the two S100 proteins is quite different.     

Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide

Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca(2+)-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.