- Assessment of S100 protein structure, function, and expression.
- Mechanism of action of S100 proteins in disease pathophysiology.
- S100 proteins as biomarkers for disease detection and prognosis.
- Therapeutic strategies targeting S100 proteins to treat disease.
Introduction
The S100 family of proteins contains 25 known members that share a high degree of sequence and structural similarity. S100 family members are multifunctional proteins that regulate a diverse array of cellular processes including proliferation, differentiation, inflammation, migration and/or invasion, apoptosis, Ca2+ homeostasis, and energy metabolism. The S100 protein family is a multigene calcium-binding family, each encoded by a separate gene. S100 proteins exert their actions usually through calcium binding, although Zn2+ and Cu2+ have also been shown to regulate their biological activity. The S100 protein family is an acidic calcium-modulated protein family of low molecular weight (10-12 kDa), mainly expressed in vertebrates. S100 proteins have the ability to form homodimers, heterodimers and oligomers. Only a limited number of family members have been characterized in depth, and the roles of other members are likely undervalued.
Biological functions of S100 proteins: a broad range of intracellular and extracellular functions. Intracellular functions include: regulation of enzyme activity and protein phosphorylation, calcium homeostasis, regulation of cytoskeletal components and of transcriptional factors. Extracellular functions of S100 proteins: act as Ca2+ sensor proteins in the cell and transmit a signal by Ca2+-dependent binding to the target protein, regulating its biological activity. Extracellularly in the presence of high concentrations of Ca2+ and Zn2+, S100 proteins can form polymers and bind to the receptor named RAGE.
S100 protein reactivity: seen in 50-90% of tumors with extent and intensity of reactivity dependent on grade of tumor.
Due to the diverse range of cellular functions undertaken by S100 proteins, some family members have been given more than one name. These include:
- S100A4 - calvasculin
- S100A6 - calcyclin
- the dimer formed by S100A8/A9 - calprotectin
- additionally, calgranulins comprise a group of S100 proteins including:
- S100A8 (calgranulin A)
- S100A9 (calgranulin B)
- S100A12 (calgranulin C)
which act as sensors of intracellular Ca2+ levels
S100 protein structure, expression, and function
Molecular structure
The S100s constitute a family of proteins where each protein is encoded by an individual gene. Of the 25 human S100 genes, 19 (group A S100 proteins) are located within chromosome 1q21. Other members (S100A11P, S100B, S100G, S100P and S100Z) map to different regions. Each member of the S100 protein family has a similar molecular mass of 10-12 KDa, and they each share 25-65% similarity in their amino acid sequence. Upon Ca2+ binding, the S100 proteins experience a conformational change that allows interaction with target proteins. The distribution of hydrophobic and charged residues, together with differences in surface configurations, contribute to the specific target binding patterns described amongst S100 family members.
Expression
Members of the S100 gene family show different patterns of both cell- and tissue-specific expression. Expression of S100 proteins is carefully regulated in order to ensure the maintenance of immune homeostasis. Calprotectin (S100A8/A9): constitutively expressed in certain immune cells (monocytes, neutrophils, dendritic cells). Upon activation, it is also expressed in fibroblasts or mature macrophages, amongst others. Epigenetic mechanisms play a vital role in S100 gene expression regulation (methylation of DNA CpG islands: a common method of transcriptional repression). DNA hypomethylation significantly induce expression of S100 members in prostate and gastric cancer.
Function
S100 proteins have been implicated in the control of a wide number of intracellular and/or extracellular functions, including regulation of cell apoptosis, proliferation, differentiation, migration/invasion, energy metabolism, Ca2+ homeostasis, protein phosphorylation and inflammation in different cell types.
S100s as damage associated molecular pattern (DAMP) molecules
DAMPs play a key role in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis, osteoarthritis and atherosclerosis. After cell damage/stress or activation of immune cells including neutrophils and macrophages, S100 proteins are released to the extracellular space where they play a key role in the regulation of several immune and inflammatory processes. They act as DAMP molecules to activate both immune and endothelial cells by binding to toll-like receptors (TLR)s and receptors for advanced-glycation end products (RAGE).
S100s in immune cell migration, invasion and differentiation
Increasing evidence shows that several S100 proteins contribute to leukocyte migration. For instance, as well as inducing pro-inflammatory cytokine production in macrophages through the activation of the NF-kB and p38 mitogen activated protein kinase (MAPK) pathways, S100A8/A9 has been seen to mediate immune cell migration; S100A12 has been shown to induce the production of pro-inflammatory cytokines interleukin (IL)-6 and -8 through RAGE-dependent NF-kB activation, resulting in the recruitment of monocytes; S100A10 has been reported to recruit macrophages to tumour sites; whereas S100A8/S100A9 have been shown to signal through RAGE to mediate the effect of TNF-a on the differentiation of myeloid-derived suppressor cells.
S100s as biomarkers for disease
Since a number of S100 proteins can be identified in body fluids, they may be used as biomarkers to detect a specific disease, where their increased expression levels are indicative of pathological conditions.
As such, S100A4 has been reported as a novel biomarker and an important regulator of glioma stem cells, with its increased expression contributing to the appearance of a metastatic phenotype, as well as having been described as a marker for lupus nephritis activity, a determinant factor for the onset of the complex inflammatory autoimmune disease lupus erythematosus; increased serum levels of S100A6 have been reported in patients with gastric cancer; S100A7 levels have been found to be increased in cerebrospinal fluid and brain of patients with Alzheimer's disease; blood levels of S100A12 are increased in patients with diabetes and it has also been used as a biomarker for detection of other inflammatory diseases such as systemic-onset juvenile idiopathic arthritis; augmented serum levels of
S100A8/A9 have been seen in individuals with obesity and in patients with coronary artery diseases. 
Importantly, S100A8/A9 has also proven to be a useful biomarker for disease activity in the management of inflammatory bowel diseases such as Crohn's disease, and faecal S100A8/A9 detection can ben used to differentiate inflammatory bowel disease from irritable bowel syndrome.
Finally, evidence showed that S100B can be used as a monitoring and prediction tool for management of traumatic brain injury, while its overexpression has also been associated with certain genetic disorders such as Down syndrome and even to certain mood disorders as a consequence of glial pathology.
S100s as therapeutic targets
S100 proteins, particularly calgranulins (S100A8, A9 and A12), play a major role in the mediation of the immune responses characteristic of a series of diseases, including inflammatory arthritis, atherosclerosis and microbial infections, as well as joint inflammation and cartilage degradation in patients with rheumatoid arthritis. S100A7 has been found to be abundantly expressed in psoriatic lesions or in serum from psoriatic patients as well as in dermatitis skin lesions, induced by pro-inflammatory factors such as TNF-a, IL-17 and IL-22.
Several S100 proteins bind to TLR4 and RAGE. Importantly, the heterodimeric form of S100A8/S100A9 can bind TLR4, whereas high extracellular Ca2+ concentrations induce the formation of S100A8/S100A9 tetramers, preventing its interaction with TLR4, thus providing an autoinhibitory mechanism for modulating S100A8/9 biological activity.
The use of S100 function-blocking antibodies might provide an effective therapeutic strategy to treat cancers and immune disorders. Anti-allergic drugs have been reported to bind to S100A12, blocking downstream RAGE signalling and subsequent NF-kB activation.
Extracellular S100A8/S100A9 levels: closely linked to inflammatory and autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis, diabetic nephropathy, cardiovascular disease). S100A8 would be a good target against obesity-induced chronic inflammation. Increased S100A8/A9 expression in the tumour microenvironment is associated with the progression and aggressiveness of the disease.
S100A4
S100A4 plays a significant role in many physiological functions including cell motility, adhesion, proliferation, invasion, and metastasis. Intracellular S100A4 binds to proteins of the cytoskeleton including F-actin and non-muscle myosin heavy chains, both involved in cellular stability and/or motility. By contrast, extracellular S100A4 regulates the expression of extracellular matrix (ECM)-remodelling enzymes such as MMPs, which are implicated in mediating cellular migration in various tissues, and can signal through membrane receptors to activate proinflammatory pathways.
S100A4 in disease
S100A4 and cancer
S100A4, together with many other proteins: involved in the complex multi-step process of cancer metastasis at the molecular level. S100A4, secreted from both tumour and non-malignant cells, plays a key role in the regulation of angiogenesis, cell migration and inflammation.
S100A4 levels are increased in many types of cancers and tumour microenvironments, including brain, breast, lung, gastric, liver, pancreatic, colorectal and prostate cancers amongst others, in addition to osteosarcoma, leukaemia and malignant melanoma, always associated to poor prognosis. S100A4 is a strong likely biomarker for cancer diagnosis and metastasis prediction.
S100A4 and non-cancer pathologies
Even though S100A4 is best known in a disease context for its participation in cancer progression and metastasis, an increase in S100A4 expression has also been associated with several non-tumour pathophysiological processes including tissue fibrosis, inflammation, neuroprotection and cardiovascular events.
S100 family signaling network and related proteins in pancreatic cancer
Multiple proteins of the S100 protein family are closely related to pancreatic cancer, including the following proteins: S100A2, S100A4, S100A6, S100P, S100A11.
S100 proteins interact with receptor for advanced glycation end-products (RAGE), p53 and p21, which play a role in the degradation of the extracellular matrix (ECM) and metastasis, and also interact with cytoskeletal proteins and the plasma membrane in pancreatic cancer progression and metastasis. S100A11 and S100P are significant tumour markers for pancreatic cancer and unfavorable predictors for the prognosis of patients who have undergone surgical resection. S100A2 has been suggested to be a negative prognosis biomarker in pancreatic cancer, and the expression of S100A6 may be an independent-prognosis impact factor. The expression of S100A4 and S100P is associated with drug resistance and differentiation, metastasis and clinical outcome. Conclusion: S100 proteins may be used as molecular markers for the early diagnosis, treatment and prognosis of pancreatic cancer.
The S100 family: intracellular and extracellular activities
Intracellular activities of S100 proteins
Members of the S100 family interact with p53 and this produces differential effects, depending on the activity of the protein involved. Both S100A4 and S100B are thought to inhibit p53 phosphorylation, leading to the inhibition of this transcriptional activity, thereby compromising p53 tumor-suppressor activity. By contrast, S100A2 promotes p53 transcriptional activity. Thus the balanced actions of different S100 proteins within a cell determine its function. Many of the S100 family members are involved in modulating cytoskeletal dynamics.
Extracellular roles of S100 proteins
S100 proteins are involved in the extracellular stimulation of neuronal survival, differentiation and astrocyte proliferation, resulting in neuronal death via apoptosis, and stimulate (in some cases) or inhibit (in other cases) the activity of inflammatory cells. S100 proteins are closely related to a variety of human diseases, such as neurological disorders, cancer, inflammation and heart disease.
S100 proteins: methods of measurement
Analytical methods such as immunoradiometric assay (IRMA), mass spectroscopy, western blot, ELISA (enzyme linked immunosorbent assay), electrochemiluminence and quantitative PCR, can detect S100 changes in immunohistochemical expression or in serum concentration with high sensitivity, providing an important tool in clinical diagnosis.
S100 expression in related diseases
Diseases associated with altered expression levels of S100 proteins can be classified into four categories:
1. Neurologic disorders
As protein S100B is primarily produced by astrocytes in CNS, its increased expression - as well as that of glial fibrillary acidic protein (GFAP) - represents a hallmark of astrocytic activation. S100B protein's autocrine effects on astrocytes (upregulation of IL-6, TNF-alpha expression) are mediated through its interaction with RAGE (Receptor for Advanced Glycation End products). Secretion of S100B is an early process during the glial response to metabolic injury (oxygen, serum and glucose deprivation). Elevated levels are observed in patients suffering from chronic neurodegenerative disorders such as Alzheimer's disease. Elevated levels of S100B originating from necrotic tissues might enhance or even amplify neurodegeneration by S100B-induced apoptosis.
It has been reported that serum and CSF S100 level is raised in systemic lupus erythematous with neuropsychiatric involvement (i.e. organic brain syndrome, seizures, cerebral vascular accident, psychosis) and in obstructive sleep apnea syndrome, reflecting the ongoing neurological damage. Significant increase of serum S100B is observed during exacerbations of bipolar disorder (episodes of mania and depression).
The above described wide range of applications has led to the consideration of S100B measurement in neurologic disorders as analogous to that of CRP in systemic inflammation.
2. Neoplastic disorders
Different forms of cancer exhibit dramatic changes in the expression of S100 proteins such as S100B, S100A2, S100A4, S100A6, and S100P. The S100-RAGE signalling pathway plays an important role in linking inflammation and cancer and in tumour cell survival and malignant progression (RAGE-deficient tumours are characterized by accelerated apoptosis, reduced activation of NFkB and significantly impaired proliferation).
Elevated levels of S100A4 (metastasin) are associated with poor survival rates in breast cancer patients. Increased serum concentration of S100A4 is also found in esophageal squamous and colon carcinoma, invasive pancreatic carcinoma, non small cell lung cancer, bladder carcinoma and correlates with a worse outcome and more aggressive disease.
Concerning the detection of brain metastases, serum concentration of S100B has a good negative predictive value. As its levels may also reflect the existence of cerebrovascular ischemic changes without infiltrating tumour, it may be used in conjunction with proApolipoprotein A1 for a sufficiently specific serum-based diagnostic of the presence of metastatic brain tumours.
S100A2 is highly expressed in tumours such as: non-small lung cancer, gastric/oesophagea squamos cancer, lymphoma, granular cell tumours of the gastrointestinal tract, renal tumours, papillary and anaplastic thyroid carcinomas.
There is accumulating evidence that BRCA1 negative breast carcinomas exhibit increased expression of S100A7.
S100A8 and S100A9 form a heterodimer complex implicated in regulating cell proliferation and in the metastatic process.
Summary of the links that have been noted between various types of cancer and members of the S100 protein family:
Cancer Members of the S100 protein family
Melanoma S100B (established use), S100A4, S100A2
Breast S100A4, S100A7 (promising results)
S100A8, S100A9, S100A2, S100A6, S100A11
Pancreatic S100A4, S100A10, S100A11, S100P (8-fold increase)
Colorectal S100A4, S100A6, S100A8, S100A9, S100A11
Gastric S100A2, S100A4, S100A8, S100A9, S100A11
Bladder S100A4, S100A11 (down-regulation associated with decreased survival)
Ovarian S100A1, S100A4
Prostate S100A2, S100A4, S100A11 (up-regulation associated with advanced stage)
Lung (squamous cell) S100A2, S100A4, S100P
Renal S100A1, S100A11,
S100A2 (3.8-fold decrease in 93% of patients)
Thyroid S100A2, S100A4
Lymphoma S100A2, S100P
3. Cardiac diseases
S100A1 is specifically and highly expressed in the mammalian myocardium, where it modulates contractile performance of the heart via interaction with contractile filaments and with proteins of the sarcoplasmic reticulum (SR). It also increases the release of calcium from the sarcoplasmic reticulum by interacting with the ryanodine receptor. S100A1 is up-regulated in right ventricular hypertrophy and down-regulated in end-stage heart failure, indicating a correlation between S100A1 expression and contractile performance.
4. Inflammatory diseases
S100A8, S100A9, and S100A12, are predominantly expressed in phagocytes and are strongly associated with pro-inflammatory functions. They are secreted especially at sites of inflammation. The serum concentrations of these S100 proteins correlate with inflammatory disease activity; high levels were identified in several inflammatory disorders such as rheumatoid arthritis, chronic bronchitis, and cystic fibrosis. S100A7, S100A8, S100A9, and S100A12 are up-regulated in active psoriatic lesions. Overexpression of S100A7 (acting as a keratinocyte- derived chemotactic agent for immune cells) is also seen in many epidermal inflammatory diseases, like atopic dermatitis, mycosis fungoides and Darier's disease. Antiallergic drugs which bind to S100A12 might block the S100 protein-RAGE interaction, implying a promising approach to anti-inflammatory therapy. Enteric glial-derived S100B (associated with the onset of inflammation) is increased in the duodenum of patients with celiac disease and contributes to nitric oxide production.
Conclusions
Characterization of primary tumor lesions has been used to identify and evaluate the risk in the development of tumor metastasis and to predict prognosis and therapy responses in various types of cancer. As a result, several S100 members, mainly S100A4 and S100A8/9, have been identified as key players in the pathogenesis of many types of cancer, as well as of several other disease conditions including diabetes and other inflammatory diseases. Elucidating the mechanisms of action of S100 proteins in the pathophysiology of these diseases may therefore lead to the development and application of novel, more effective therapeutic approaches. S100 proteins can be used as biomarkers in early disease detection and prognosis, and in the development of novel strategies based around anti-S100 therapies.
The members of the S100 protein family, through their interaction with several effector proteins, are involved in the regulation of a diverse spectrum of cellular processes. Although their pathophysiologic implications still require further clarification, some of these proteins have already been successfully investigated in clinical context.
Members of the S100 protein family proved to be useful biomarkers in clinical applications and the S100 protein-targeted therapies emerge as useful opportunities in specific clinical settings.
