Interaction between S100P and the anti-allergy drug cromolyn

The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling is involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex.     

Structural Insights into Calcium-Bound S100P and the V Domain of the RAGE Complex

The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE) and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca²⁺-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC), fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca⁺²-bound S100P was found to lie in the micromolar range (K_d of ∼6 µM). NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.     

Immunohistochemical Expression of RAGE and Its Ligand (S100A9) in Cervical Lesions

Altered expressions of receptor for advanced glycation end-products (RAGE) and its ligand (S100A9) are observed in many cancers and play a key role in inflammation-associated cancer. In our previous study, by two-dimensional gel electrophoresis followed by mass spectrometry, the expression of S100A9 protein was found to increase in squamous cervical cancer compared with adjacent normal cervical tissues. Therefore, in the present study we observed the expressions of S100A9 and RAGE in 30 chronic cervicitis, 50 cervical intraepithelial neoplasia (CIN), and 40 squamous cervical cancer (SCC) using immunohistochemical analysis and analyzed the differential expression and possible role of S100A9 and RAGE in cancer development. Immunohistochemical findings were as follows: the expressions of S100A9 and RAGE were demonstrated in chronic cervicitis, CIN, and SCC. Moreover, their expressions were gradually increasing as the tumor progressed. In SCC, the staining scores of S100A9 and RAGE were significantly higher in well-differentiated tumors compared to moderately and poorly differentiated tumors. The expression of S100A9 in epithelial cells exhibited a positive correlation to RAGE expression in chronic cervicitis, CIN, and SCC. There were no significant difference of S100A9 immunoreactivity in stromal cells among chronic cervicitis, CIN, and SCC. Moreover, there was no correlation between S100A9 immunoreactivity in stromal cells of SCC and clinicopathological parameters. Finally, double immunohistochemistry illustrated that RAGE and S100A9 co-express in SCC. In conclusion, RAGE binds its ligand (S100A9), which plays an important role in the development of SCC. In addition, the expressions of S100A9 and RAGE in SCC tumor cells were closely associated with histological differentiation.     

Targeting S100P inhibits colon cancer growth and metastasis by Lentivirus-mediated RNA interference and proteomic analysis

S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and β-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis.     

S100P stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE)

S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-kappaB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival.     

RAGE and RAGE ligands in cancer

The receptor for advanced glycation end-products (RAGE) is a multifunctional receptor with multiple ligands that is known to play a key role in several diseases, including diabetes, arthritis, and Alzheimer's disease. Recent evidence indicates that this receptor also has an important role in cancer. RAGE ligands, which include the S100/calgranulins and high-mobility group box 1 (HMGB1) ligands, are expressed and secreted by cancer cells and are associated with increased metastasis and poorer outcomes in a wide variety of tumors. These ligands can interact in an autocrine manner to directly activate cancer cells and stimulate proliferation, invasion, chemoresistance, and metastasis. RAGE ligands derived from cancer cells can also influence a variety of important cell types within the tumor microenvironment, including fibroblasts, leukocytes, and vascular cells, leading to increased fibrosis, inflammation, and angiogenesis. Several of the cells in the tumor microenvironment also produce RAGE ligands. Most of the cancer-promoting effects of RAGE ligands are the result of their interaction with RAGE. However, these ligands also often have separate intracellular roles, and some may interact with other extracellular targets, so it is not currently possible to assign all of their effects to RAGE activation. Despite these complications, the bulk of the evidence supports the premise that the ligand-RAGE axis is an important target for therapeutic intervention in cancer.     

Stem cells in the etiology and treatment of cancer

Using approaches first applied in human leukemias, recent progress has been made in the identification of putative cancer stem cells in several different carcinomas and other solid cancers. Additional studies have suggested that cancer stem cells may be derived not only from transformation of quiescent, long-term stem cells but also from short-lived progenitors that then obtain the ability to undergo self-renewal. Therefore, the heterogeneity observed in many types of human cancers may reflect the activation of specific oncogenes and/or loss of specific tumor suppressor genes and the different stem and/or progenitor cell populations in which these genetic or epigenetic events occur. Similarities have been observed in the pathways regulating stem cell homing and metastasis, and increasing evidence also suggests that treatment failure and the recurrence of human cancer may reflect the intrinsic quiescence and drug resistance of cancer stem cells.     

Knockdown of RAGE Inhibits Growth and Invasion of Gastric Cancer Cells

The receptor for advanced glycation end-products (RAGE) is an oncogenic trans-membranous receptor, which is overexpressed in multiple human cancers. However, the role of RAGE in gastric cancer is still elusive. In this study, we investigated the expression and molecular mechanisms of RAGE in gastric cancer cells. Forty cases of gastric cancer and corresponding adjacent non-cancerous tissues (ANCT) were collected, and the expression of RAGE was assessed using immunohistochemistry (IHC) in biopsy samples. Furthermore, RAGE signaling was blocked by constructed recombinant small hairpin RNA lentiviral vector (Lv-shRAGE) used to transfect into human gastric cancer SGC-7901 cells. The expression of AKT, proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase-2 (MMP-2) was detected by Real-time PCR and Western blot assays. Cell proliferative activities and invasive capability were respectively determined by MTT and Transwell assays. Cell apoptosis and cycle distribution were analyzed by flow cytometry. As a consequence, RAGE was found highly expressed in cancer tissues compared with the ANCT (70.0% vs 45.0%, P=0.039), and correlated with lymph node metastases (P=0.026). Knockdown of RAGE reduced cell proliferation and invasion of gastric cancer with decreased expression of AKT, PCNA and MMP-2, and induced cell apoptosis and cycle arrest. Altogether, upregulation of RAGE expression is associated with lymph node metastases of gastric cancer, and blockade of RAGE signaling suppresses growth and invasion of gastric cancer cells through AKT pathway, suggesting that RAGE may represent a potential therapeutic target for this aggressive malignancy.Key words: RAGE, gastric cancer, growth, invasion     

RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins

The S100 protein family comprises at least 25 members which, with the exception of S100G, act as Ca2+-sensor proteins that participate in Ca2+ signal transduction by interacting with target proteins thereby modifying their activities. S100 proteins are expressed in vertebrates exclusively, display a cell-specific distribution, and regulate a large variety of intracellular activities. Some S100 proteins are released by a non-classical pathway and exert regulatory effects on several cell types. The receptor for advanced glycation end products (RAGE) has been shown to transduce extracellular effects of S100B, S100A4, S100A6, S100A11, S100A12, S100A13 and S100P. However, some S100 proteins can signal by engaging RAGE as well as non-RAGE receptors. Immune cells (i.e., monocytes/macrophages/microglia, neutrophils and lymphocytes), activated endothelial and vascular smooth muscle cells, neurons, astrocytes, chondrocytes and pancreatic tumor cells are the cell types reported to respond to certain S100 proteins via RAGE engagement. In general, relatively high concentrations of S100 proteins are required for activation of RAGE in responsive cells. S100B is unique in that it can engage RAGE in neurons at low and high concentrations with trophic and toxic effects, respectively, and S100A4 stimulates matrix metalloproteinase 13 release from chondrocytes at nanomolar doses in a RAGE-mediated manner. Oligomerization of S100 proteins under the non-reducing, high-Ca2+ conditions found extracellularly appears to play a relevant role in RAGE activation, and binding of at least S100A12 and S100B results in RAGE oligomerization. Thus, S100/RAGE interactions might have important consequences during development and in tissue homeostasis as well as in inflammatory, degenerative and tumor processes.     

Targeting signal transduction pathways to eliminate chemotherapeutic drug resistance and cancer stem cells

Breast cancer is one of the most common cancers and affects nearly 1 in 7 women. We have demonstrated that targeting the CaM-K, Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be a novel approach to treat drug resistant breast cancer and eliminate cancer stem cells. Common chemotherapeutic drugs, such as doxorubicin, induce the CaM-K pathway which in turn, leads to activation of anti-apoptotic pathways such as Raf/MEK/ERK and PI3K/Akt. Some drug resistant breast cancers exhibited increased expression of CaM-KIV. CaM-K inhibitors synergized with doxorubicin to induce the death of all drug resistant breast cancers examined. Since CaM-Ks are known to result in activation of the Raf/MEK/ERK and PI3K/Akt pathways, we investigated the roles that these pathways exert in breast cancer drug resistance. CaM-K inhibitors suppressed ERK activation in response to doxorubicin in both drug sensitive and resistant cells. CaM-K inhibitors also suppressed ERK activation in response to FBS in the drug resistant cells suggesting dependence on the CaM-K pathway for proliferation. Both the Raf/MEK/ERK and PI3K/Akt pathways are involved in breast cancer drug resistance as they were detected at elevated, activated levels in the drug resistant cells and introduction of constitutively activated forms of Raf-1 and Akt-1 resulted in drug resistance. Drug resistant CICs were often hypersensitive to MEK and mTOR inhibitors, implicating important roles of these pathways in drug resistance. In summary, targeting these pathways may enhance therapy of drug resistant breast cancer and eliminate CICs.Breast cancer therapy is often limited by the occurrence of drug resistance which may be due to the re-emergence of CICs. The studies outlined in this proposal may identify a potentially novel role for CaM-Ks in drug resistance and metastasis and may lead to improved approaches to treat breast tumors by eliminating CICs. Our proposed studies are highly innovative as we will determine the involvement of the CaM-K pathway in breast cancer drug resistance, metastasis and CIC formation. Similar approaches have not been previously performed. Our studies may result in the discovery of novel methods to treat breast cancer by targeting the CaM-K pathway in combination with currently used and approved chemotherapeutic regimens to eliminate CICs which may be responsible for both drug resistance and metastasis.     

S100A8 Contributes to Drug Resistance by Promoting Autophagy in Leukemia Cells

Autophagy is a double-edged sword in tumorigenesis and plays an important role in the resistance of cancer cells to chemotherapy. S100A8 is a member of the S100 calcium-binding protein family and plays an important role in the drug resistance of leukemia cells, with the mechanisms largely unknown. Here we report that S100A8 contributes to drug resistance in leukemia by promoting autophagy. S100A8 level was elevated in drug resistance leukemia cell lines relative to the nondrug resistant cell lines. Adriamycin and vincristine increased S100A8 in human leukemia cells, accompanied with upregulation of autophagy. RNA interference-mediated knockdown of S100A8 restored the chemosensitivity of leukemia cells, while overexpression of S100A8 enhanced drug resistance and increased autophagy. S100A8 physically interacted with the autophagy regulator BECN1 and was required for the formation of the BECN1-PI3KC3 complex. In addition, interaction between S100A8 and BECN1 relied upon the autophagic complex ULK1-mAtg13. Furthermore, we discovered that exogenous S100A8 induced autophagy, and RAGE was involved in exogenous S100A8-regulated autophagy. Our data demonstrated that S100A8 is involved in the development of chemoresistance in leukemia cells by regulating autophagy, and suggest that S100A8 may be a novel target for improving leukemia therapy.     

Overview of CD24 as a new molecular marker in ovarian cancer

Ovarian cancer (OC) is the fifth leading cause of cancer-related death among women. The high mortality rate is due to lack of early symptoms, late diagnosis, limited treatment options, and also emerging of drug resistance. Todays, molecular markers have become promising in tumor-targeted therapy. Several molecular markers have been known in OC immunotherapy. Identification of the specific molecular markers with prognostic significance is interested. CD24 is a small sialoglycoprotein which is localized in lipid rafts through its glycosylphosphatidylinositol (GPI) anchor. It has been reported that CD24 is overexpressed in many cancers including OC. Also, CD24 is identified as a cancer stem cell marker in OC. The CD24 expression is associated with the development, invasion, and metastasis of cancer cells. The exact role of CD24 in cancer cells is not clearly understood. Recently, CD24 has been identified as an independent prognostic marker of survival in patients with OC. In this study, we reviewed the molecular targets in OC immune-targeted therapy and also presented an overview of the new molecular marker CD24 and its association with the OC by reviewing the recent literature.     

S100A14 stimulates cell proliferation and induces cell apoptosis at different concentrations via receptor for advanced glycation end products (RAGE)

S100A14 is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effects on different types of cells. However, exact extracellular roles of S100A14 have not been clarified yet. Here we investigated the effects of S100A14 on esophageal squamous cell carcinoma (ESCC) cell lines. Results demonstrated that low doses of extracellular S100A14 stimulate cell proliferation and promote survival in KYSE180 cells through activating ERK1/2 MAPK and NF-κB signaling pathways. Immunoprecipitation assay showed that S100A14 binds to receptor for advanced glycation end products (RAGE) in KYSE180 cells. Inhibition of RAGE signaling by different approaches including siRNA for RAGE, overexpression of a dominant-negative RAGE construct or a RAGE antagonist peptide (AmphP) significantly blocked S100A14-induced effects, suggesting that S100A14 acts via RAGE ligation. Furthermore, mutation of the N-EF hand of S100A14 (E39A, E45A) virtually reduced 10 μg/ml S100A14-induced cell proliferation and ERK1/2 activation. However, high dose (80 μg/ml) of S100A14 causes apoptosis via the mitochondrial pathway with activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase. High dose S100A14 induces cell apoptosis is partially in a RAGE-dependent manner. This is the first study to demonstrate that S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades, promoting cell proliferation or triggering cell apoptosis at different doses.     

S100 to receptor for advanced glycation end-products binding assay: Looking for inhibitors

Secreted by tumor and stromal cells, S100 proteins exert their biological functions via the interaction with surface receptors. The most described receptor is the receptor for advanced glycation end-products (RAGE), thereby participating in the S100-dependent cell migration, invasion, tumor growth, angiogenesis and metastasis. Several approaches have been described for determining this interaction. Here we describe an easy, specific and highly reproducible ELISA-based method, by optimizing several parameters such as the binding and blocking buffer, interaction time and concentrations, directed to screen chemical and biological inhibitors of this interaction for S100A4, S100A7 and S100P proteins. The efficiency of the protocol was validated by using well described neutralizing agents of the RAGE receptor and of the S100A4 activity. The methodology described here will allow future works with other members of the S100 protein family and their receptors.     

RAGE and its ligands in retinal disease

RAGE, the receptor for advanced glycation endproducts (AGEs), is a multiligand signal transduction receptor of the immunoglobulin superfamily of cell surface molecules that has been implicated in the pathogenesis of diabetic complications, neurodegenerative diseases, inflammatory disorders, and cancer. These diverse biologic disorders reflect the multiplicity of ligands capable of cellular interaction via RAGE that include, in addition to AGEs, amyloid-beta (Abeta) peptide, the S100/calgranulin family of proinflammatory cytokines, and amphoterin, a member of the High Mobility Group Box (HMGB) DNA-binding proteins. In the retina, RAGE expression is present in neural cells, the vasculature, and RPE cells, and it has also been detected in pathologic cellular retinal responses including epiretinal and neovascular membrane formation. Ligands for RAGE, in particular AGEs, have emerged as relevant to the pathogenesis of diabetic retinopathy and age-related macular disease. While the understanding of RAGE and its role in retinal dysfunction with aging, diabetes mellitus, and/or activation of pro-inflammatory pathways is less complete compared to other organ systems, increasing evidence indicates that RAGE can initiate and sustain significant cellular perturbations in the inner and outer retina. For these reasons, antagonism of RAGE interactions with its ligands may be a worthwhile therapeutic target in such seemingly disparate, visually threatening retinal diseases as diabetic retinopathy, age-related macular degeneration, and proliferative vitreoretinopathy.     

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.