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.     

S100A1 and S100B interactions with annexins

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.     

S100A7, S100A10, and S100A11 are transglutaminase substrates

S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.     

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.     

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.     

S100A8 and S100A9—oxidant scavengers in inflammation

S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys₄₂ residue. Met₇₈(O) and Trp₅₄(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp₅₄(+16). Evidence for oxidation of the single Cys₃ residue in recS100A9 HOCl was weak; Met₆₃, Met₈₁, Met₈₃, and Met₉₄ were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met₆₃, Met₈₁, and Met₉₄ were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

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.     

S100 protein and down syndrome

S100 protein is a low molecular weight calcium-binding protein widely distributed in the central nervous system of vertebrates. Recent evidence suggests that S100 protein may play a role in the regulation of glial proliferation and neuronal differentiation. The gene for S100 protein has been mapped to the 21q22 region, a chromosomal locus whose duplication has been implicated in the generation of Down Syndrome (DS). This raises the possibility that abnormalities in S100 protein gene dosage at a critical period during development may be responsible for some of the neurologic abnormalities associated with DS.     

Bovine Brain S100 Proteins: Separation and Characterization of a New S100 Protein Species

Three S100 protein species (S100a, S100b, S100a') have been purified from bovine brain using a modification of standard preparative methods. A higher yield for each protein was obtained at the last separation step. Characterization by urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis, UV absorption spectra, and fluorescence parameters provided evidence of a new tryptophan-containing S100 protein called S100a', which exhibits, as S100a and S100b, the properties of a Ca2+ binding protein.     

S100-annexin complexes--structural insights

Annexins and S100 proteins represent two large, but distinct, calcium-binding protein families. Annexins are made up of a highly alpha-helical core domain that binds calcium ions, allowing them to interact with phospholipid membranes. Furthermore, some annexins, such as annexins A1 and A2, contain an N-terminal region that is expelled from the core domain on calcium binding. These events allow for the interaction of the annexin N-terminus with target proteins, such as S100. In addition, when an S100 protein binds calcium ions, it undergoes a structural reorientation of its helices, exposing a hydrophobic patch capable of interacting with its targets, including the N-terminal sequences of annexins. Structural studies of the complexes between members of these two families have revealed valuable details regarding the mechanisms of the interactions, including the binding surfaces and conformation of the annexin N-terminus. However, other S100-annexin interactions, such as those between S100A11 and annexin A6, or between dicalcin and annexins A1, A2 and A5, appear to be more complicated, involving the annexin core region, perhaps in concert with the N-terminus. The diversity of these interactions indicates that multiple forms of recognition exist between S100 proteins and annexins. S100-annexin interactions have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein. The structures and differential interactions of S100-annexin complexes may indicate that this process has several possible modes of protein-protein recognition.     

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.     

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.