S100A6 belongs to the S100 family of Ca2+-binding proteins of the EF-hand type (reviewed in Donato et al. 2013). As a member of the A group of S100 proteins, human S100A6 gene maps to chromosome 1q21. S100A6 is expressed as an 89-amino-acid protein in mouse and rat and a 90-amino-acid protein in human and rabbit. Two chicken isoforms, A (92 amino acids) and B (91 amino acids), probably result from alternative mRNA splicing. S100A6 from all these sources differs in only a few amino acids and in the length of the carboxy terminus. The S100A6 gene was initially identified in growth-arrested rodent fibroblasts stimulated with serum and suggested to have a role in cell-cycle progression as inferred by its upregulation in several tumors (reviewed in Leśniak et al. 2009). S100A6 was found to interact with calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) (Leśniak et al. 2009). Because CacyBP/SIP is a component of the ubiquitin ligase complexes, S100A6 was suggested to be involved in the ubiquitination of β-catenin (Leśniak et al. 2009), thus supporting the possibility that S100A6 might play a role in the control of cell cycle progression. S100A6’s ability to inhibit the interaction between the heat shock proteins (Hsp70 and Hsp90) and Sgt1 or Hop (Leśniak et al. 2009) suggested a potential role for S100A6 in the cellular response to different stress factors. In this respect, S100A6 was found to favor apoptosis in some cell types but to limit it in others (Leśniak et al. 2009). The in vitro interaction of S100A6 with caldesmon, calponin, tropomyosin, and kinesin light chain (Leśniak et al. 2009) suggested that S100A6 might be involved in the regulation of cytoskeleton dynamics, particularly microfilament dynamics, and in vesicular transport (Leśniak et al. 2009). As an extracellular factor, S100A6 was shown to be involved in the release of lactogen-II, insulin, and histamine (Leśniak et al. 2009). By binding to the transmembrane receptor for advanced glycation endproducts (RAGE), S100A6 induced neuronal apoptosis by causing reactive oxygen species (ROS)-dependent activation of JNK and of caspases 3 and 7 (Leśniak et al. 2009). RAGE transduces extracellular effects of several S100 proteins (Donato et al. 2013). Integrin β1 is another potential membrane protein transducing extracellular effects of S100A6 (Jurewicz et al. 2014).
Regulation of Expression
Several factors have been shown to increase S100A6 mRNA and protein levels such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), serum, retinoic acid, estrogen, palmitate, vasopressin, and gastrin (Leśniak et al. 2009) (Fig. 1). S100A6 levels are also upregulated upon stress conditions such as ischemia, mechanical force, irradiation, and oxidative stress (Leśniak et al. 2009). In vivo, S100A6 protein levels are elevated in myocardial disease and in many types of tumor cells (see below). However, the primary cause(s) of this increase remain(s) to be fully elucidated. A decrease in S100A6 levels was observed during the course of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)- and etoposid-induced apoptosis in human breast cancer cells (Leśniak et al. 2009). At the transcriptional level, USF (upstream transcription factor), NF-κB (nuclear transcription factor κB), Sp1 (specificity protein 1), and Nrf2 (Nf-E2 related factor 2) have been shown to activate the S100A6 gene promoter (Leśniak et al. 2009), whereas the tumor suppressor, p53, acts indirectly to suppress transcription via interference with Sp1 and with NFκB function on the S100A6 promoter (Leśniak et al. 2009) (Fig. 2). Insufficient suppression of S100A6 gene by p53 mutants might thus be responsible for S100A6 overexpression and cell cycle deregulation in cancer tissues.
Regulation of Activity
Similar to other S100 proteins, S100A6 forms homodimers in solution in the absence of Ca2+ (Otterbein et al. 2002) (Fig. 3). Each S100A6 subunit binds two calcium ions. Ca2+-binding induces modest conformational changes in each subunit of the S100A6 homodimer (Otterbein et al. 2002) as observed for other S100 proteins (Donato et al. 2013). The Ca2+ binding causes hydrophobic residues of helices III and IV of each subunit to become exposed which enables S100A6 to interact with many target proteins such as glyceraldehyde-3-phosphate dehydrogenase, annexin II, annexin XI, annexin VI, and tropomyosin (Leśniak et al. 2009) (Fig. 4). Additional intracellular interacting proteins are caldesmon, calponin, and lysozyme; CacyBP/SIP, Sgt1, and melusin; p53 and the Hsp90/Hsp70-organizing protein (Hop); and kinesin light chain (Leśniak et al. 2009) (Fig. 4). A Ca2+- or Zn2+-dependent S100A6/S100B heterodimer was identified in a yeast two-hybrid assay and confirmed in vivo (Leśniak et al. 2009). However, no functional correlates have been reported for these interactions with the exception of CacyBP/SIP (see section “S100A6 and Cell Proliferation and Cancer”). S100A6 also binds Zn2+ (Leśniak et al. 2009). The binding of Zn2+ induces conformational changes in the S100A6 molecule that are different from those observed following Ca2+ binding. At present, there are no data showing a potential zinc-dependent activity of S100A6.
S100A6 and Cell Proliferation and Cancer
S100A6 affects cell proliferation and cancer development by acting both from within and from outside cells. S100A6 is overexpressed in breast, stomach, pancreas, and colon cancer and in melanoma, whereas it is underexpressed in prostate and oral cancer (reviewed in Chen et al. 2014). It is considered as a diagnostic marker or prognostic factor in pancreatic cancer, gastric cancer, prostate cancer, melanoma, non–small cell lung carcinoma, and hepatocellular carcinoma. S100A6 affects murine models of cancer; however, its contribution to promoting a cancerous phenotype has only been examined in a limited number of model systems and the mechanistic basis for the observed effects on tumor progression has not been fully delineated. In gastric cancer cells, S100A6 negatively regulates its partner-CacyBP/SIP mediated inhibition of cell proliferation and tumorigenesis by affecting β-catenin degradation (Chen et al. 2014); blockade of CacyBP/SIP by S100A6 results in reduced levels of β-catenin degradation (Fig. 5). Also, S100A6 enhanced migration and invasion of pancreatic ductal adenocarcinoma cells and promoted epithelial-mesenchymal transition via activation of β-catenin (Chen et al. 2015) (Fig. 5). The tumorigenic activity of overexpressed S100A6 was reported for clear cell renal cell carcinoma in which the protein inhibited apoptosis and the expression of the antitumor chemokine, CXCL14 (Lyu et al. 2015). Additional evidence for S100A6-CacyBP/SIP interactions comes from the observation that S100A6 inhibits CacyBP/SIP phosphorylation by casein kinase II similar to the CacyBP/SIP phosphorylation inhibitor, DRB, which results in reduced phosphatase CacyBP/SIP activity towards the mitogen-activated protein kinases (MAPKs), ERK1/2 (Wasik et al. 2016) (Fig. 5), which in turn might sustain cell proliferation and/or tumorigenesis.
S100A6 is reported to regulate endothelial cell cycle and senescence. In primary human endothelial cells, depletion of S100A6 caused increased cell-cycle arrest in G2/M phase. S100A6 depletion caused a decrease in both cyclin-dependent kinase (CDK1), phosphorylated CDK1 levels, cyclin A1 and cyclin B genes with effects on cell-cycle progression (Chen et al. 2014) (Fig. 6). A role for S100A6 as an intracellular regulator of cell proliferation and differentiation is suggested by the finding that S100A6 becomes downregulated at the beginning of keratinocyte differentiation and that S100A6 overexpression in these cells causes accelerated proliferation, enhanced adhesive properties, and reduced differentiation (Graczyk and Leśniak 2014) (Fig. 7).
Transfection with recombinant S100A6 of or administration of recombinant S100A6 protein to HCT116, a colorectal carcinoma cell line with relative low S100A6 expression, resulted in enhanced cell proliferation and migration, MAPK activation in vitro, and tumor growth in vivo (Chen et al. 2014) (Fig. 8). Conversely, RNAi-mediated knockdown of S100A6 in LoVo, a colorectal carcinoma cell line with relative high S100A6 expression, resulted in reduced cell proliferation, migration, and MAPK activity. S100A6-induced proliferation was partially attenuated by an ERK1/2 inhibitor while migration was suppressed by a p38 MAPK inhibitor (Chen et al. 2014). These results suggest that S100A6 might act as an extracellular signaling molecule affecting cancer cells in a receptor-mediated manner. One study reported that S100A6 binds the C2 domain of RAGE (reviewed in Donato et al. 2013), whereas another study showed that S100A6 binds RAGE V domain similar to other S100 proteins (Mohan et al. 2013) (Fig. 9). S100A6 increases adhesion and inhibits proliferation of mesenchymal stem cells isolated from Wharton’s jelly of the umbilical cord (Jurewicz et al. 2014). Integrin β1 appears to be the membrane protein (receptor) transducing these S100A6 effects because neutralization of integrin β1 but not RAGE blunted S100A6’s effects in mesenchymal stem cells (Fig. 10). There are several examples of interaction of S100 proteins with receptors other than RAGE (Donato et al. 2013). On the other hand, exogenous expression of S100A6 in mesenchymal stem cells increased proliferation and inhibited osteogenic differentiation and stimulated osteosarcoma growth in vivo (Li et al. 2015).
S100A6 modulates RAGE-dependent survival of neuroblastoma cells by triggering apoptosis and generation of ROS through c-Jun NH2 terminal protein kinase activation (Donato et al. 2013). S100A6 may regulate secretory processes in some cells. It stimulates secretion of lactogen II by trophoblasts and insulin release from pancreatic islet cells. S100A6 may modulate allergic responses by inhibiting histamine release by mast cells (Leśniak et al. 2009).
The role of S100A6 as an intracellular regulator of cell proliferation/apoptosis is further complicated by its effects on the antitumor, p53 (Graczyk et al. 2013), and by the finding that p53 acts indirectly to suppress S100A6 transcription (Leśniak et al. 2009) (Fig. 2). S100A6 competes with MDM2, a ubiquitin E3 ligase that degrades p53, and with p300 acetyltransferase for binding to p53 (Graczyk et al. 2013) (Fig. 11). Once acetylated, p53 loses the ability to bind S100A6, suggesting that high S100A6 concentrations might interfere with p53 acetylation and, thus, that S100A6 might protect p53 against untimely degradation and/or acetylation thus resulting in the promotion of p53 nuclear translocation and, likely, p53 transcriptional activity. In this perspective, S100A6 might aid in cell proliferation arrest and/or apoptosis. However, the opposite has also been observed in mixed-lineage leukemia/AF4-positive acute lymphoblastic leukemia where IL-24-iduced inhibition of S100A6 expression was shown to exert proapoptotic effects, which points to an antiapoptotic role of S100A6 in these cells (Tamai et al. 2014 and Refs. therein) (Fig. 12). For S100A6’s antiapoptotic effects also see section “S100A6 and Stem Cells” below.
S100A6 and Cytoskeleton
Intracellular S100A6 has been functionally linked to changes in cellular motility and cytoskeletal reorganization, but a clear mechanistic picture is still lacking (Leśniak et al. 2009). Knockdown of S100A6 in NIH-3T3 fibroblastic cells causes a reorganization of the actin cytoskeleton with an extensive cortical network of actin filaments and tropomyosin structures and increase in the number of focal adhesions at the cell periphery. Thus, S100A6 effects on actin filaments and tropomyosin structures might be responsible at least in part for the large increase in lamellipodia and possibly for the enhancement in cellular motility seen when S100A6 levels are decreased by, e.g., siRNA techniques. The involvement of S100A6 in the motility of cancer cells has also been reported, albeit with contradictory results. Down- or upregulating of S100A6 expression in osteosarcoma cells led to increased or decreased migration, respectively, suggesting a role for S100A6 as an inhibitor of cell motility in cultured cells. However, S100A6 has also been shown to promote cellular motility in pancreatic cancer cells by a mechanism that is dependent on the presence of annexin 2. Elevated levels of intracellular S100A6 have been shown to be associated with tumorigenesis (Leśniak et al. 2009) and the ability of colorectal adenocarcinoma cells and Ras-transformed NIH 3T3 cells to metastasize to form secondary lesions. Yet, the molecular mechanisms underpinning S100A6’s ability to regulate cell motility have remained elusive. Direct interaction between S100A6 and the tropomyosin-actin complex has been shown in vitro but remains to be confirmed in vivo; the only current evidence suggests that S100A6 acts as a downregulator of tropomyosin expression. S100A6 interacts in vitro with other components of the actin cytoskeletal architecture, such as the myosin ATPase inhibitors, caldesmon, and calponin, but no mechanistic link to cell motility has been demonstrated.
S100A6 and Stem Cells
S100A6 was shown to be expressed in neural stem cells in the subgranular zone of the dentate gyrus in adult hippocampus (Yamada and Jinno 2014) – a major neurogenic niche. These S100A6-expressing cells were recognized as astrocyte precursors. The finding that S100A6 was not detected in mature astrocytes suggested that S100A6 might play an important, yet unknown role during astrocytic differentiation of neural stem cells (Fig. 7). Possibly, S100A6 has to be downregulated for astrocyte precursors to undergo differentiation, as observed for keratinocyte differentiation (Graczyk and Leśniak 2014). S100A6 also marks glial precursor cells in neuroblastoma. S100A6 expression is increased in the periinfarct zone of rat heart postinfarction and functions as a global negative regulator of the induction of cardiac genes by trophic stimuli (Tsoporis et al. 2005) (Fig. 13). S100A6 is induced in cardiomyocytes by TNF-α via NF-κB activation and protects cardiomyocytes from TNF-α-induced apoptosis by associating with p53 and interfering with p53 phosphorylation(Leśniak et al. 2009).
S100A6 and Neurodegenerative Diseases
In Alzheimer’s disease mouse models, astrocytic S100A6 protein was shown to be homogeneously upregulated within the white matter, whereas within the gray matter almost all S100A6 immunoreactivity was found to be concentrated in astrocytes surrounding the Aβ amyloid deposits of senile plaques (Leśniak and Słomnicki 2009). S100A6 is also overexpressed in astrocytes located near impaired axons of motoneurons in amyotrophic lateral sclerosis (ALS) (Leśniak et al. 2009). These findings suggests that S100A6 might participate in the pathophysiology of Alzheimer’s disease and ALS, respectively. Mechanistically, S100A6 was shown to form oligomers and amyloid-like fibrils, an event negatively regulated by Ca2+, and to potentiate in vitro the aggregation of superoxide dismutase-1 (SOD1) (Botelho et al. 2012) that forms cytoplasmic aggregates in ALS-affected neurons. Although S100A6 oligomers but not fibrils proved toxic to neuronal cell in culture (Botelho et al. 2012), there are no data to demonstrate that S100A6 plays a role in the promotion of SOD1 aggregates in ALS neurons.
S100A6 as a Serum Marker of Disease
Serum levels of S100A6 are significantly elevated in early stage non–small cell lung cancer (Chen et al. 2014; Wang et al. 2016), gastric cancer (Zhang et al. 2014), urinary bladder urothelial carcinoma (Nishi et al. 2014), and ovarian cancer (Chen et al. 2014) as well as in acute coronary syndrome and myocardial infarction (Cai et al. 2011).
S100A6 protein belongs to the A group of the S100 protein family of Ca2+-binding proteins. Its expression is restricted to a limited number of cell types in adult normal tissues and in several tumor cell types. As an intracellular protein, S100A6 has been implicated in the regulation of several cellular functions such as proliferation, apoptosis, the cytoskeleton dynamics, and the cellular response to different stress factors. However, functional studies are scarce. Studies of S100A6’s interaction with and inhibition of its partner, CacyBP/SIP − an inhibitor of cell proliferation and tumorigenesis by virtue of its ability to promote degradation of β-catenin – support a role for S100A6 as a positive regulator of cell proliferation in the epidermis and tumor cells and an antiapoptotic factor in certain leukemias. Also, upregulation of S100A6 in periinfarct cardiomyocytes results in reduction of p53-induced apoptosis via interference with p53 phosphorylation and inhibition of induction of fetal genes responsible for cardiomyocytes hypertrophy. On the other hand, interaction with the tumor suppressor, p53, implicates S100A6 in apoptosis, with high concentrations of S100A6, as is typical of certain tumor cells, protecting p53 from inactivation by p300 acetyltransferase and degradation by MDM2. S100A6 can be secreted/released by certain cell types which points to extracellular effects of the protein. RAGE and integrin β1 might transduce extracellular S100A6’s effects, but further analyses in physiological and pathological contexts are required. Lastly, dosage of serum S100A6 might aid in diagnosis in oncology.
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