"There is only one motive fun." Perspectives of participants and providers of physical exercise for people with Parkinson's disease.

PURPOSE: To explore the perspectives of people with Parkinson's disease (PD) and exercise providers regarding facilitating factors, barriers, needs, and demands relating to physical exercise for people with PD. MATERIALS AND METHODS: Focus group discussions or telephone interviews of 30 people with PD (with or without an active sports history) and 13 providers were conducted and analyzed using structuring content analysis. RESULTS: Factors facilitating participation in physical exercise included motivation-enhancing elements (enjoyment, group training environment) and providers with sufficient qualifications in PD-specific training demands. Identified barriers were lack of motivation, physical limitations, poor service accessibility, and inadequate matching of intervention groups based on capability or age. Providers found it difficult to design and conduct group trainings for people with PD with varying physical limitations. Having an active sports history before PD-onset was described as generally beneficial, though a competitive mindset could lead to frustration. People with PD reported needing their physicians to provide better education regarding physical exercise. CONCLUSION: Enjoyment of physical exercise is a key aspect of maintaining physical activity engagement, which should be considered more in research and clinical practice. Developing qualifications for providers could help to broaden and enhance the dissemination of PD-specific exercise approaches. Physicians should be trained to encourage physical exercise.Implications for rehabilitationPhysicians should highlight the benefits and be knowledgeable regarding the availability of physical exercise interventions for people with PD.Additional physical exercise providers should become qualified to work with people with PD.The joyfulness of physical exercise interventions is a key aspect of maintaining physical activity engagement for people with PD.

S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin.

OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-ß/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

Effect of Anti-S100A4 Monoclonal Antibody Treatment on Experimental Skin Fibrosis and Systemic Sclerosis-Specific Transcriptional Signatures in Human Skin.

OBJECTIVE: S100A4 is a DAMP protein. S100A4 is overexpressed in patients with systemic sclerosis (SSc), and levels correlate with organ involvement and disease activity. S100A4-/- mice are protected from fibrosis. The aim of this study was to assess the antifibrotic effects of anti-S100A4 monoclonal antibody (mAb) in murine models of SSc and in precision cut skin slices of patients with SSc. METHODS: The effects of anti-S100A4 mAbs were evaluated in a bleomycin-induced skin fibrosis model and in Tsk-1 mice with a therapeutic dosing regimen. In addition, the effects of anti-S100A4 mAbs on precision cut SSc skin slices were analyzed by RNA sequencing. RESULTS: Inhibition of S100A4 was effective in the treatment of pre-established bleomycin-induced skin fibrosis and in regression of pre-established fibrosis with reduced dermal thickening, myofibroblast counts, and collagen accumulation. Transcriptional profiling demonstrated targeting of multiple profibrotic and proinflammatory processes relevant to the pathogenesis of SSc on targeted S100A4 inhibition in a bleomycin-induced skin fibrosis model. Moreover, targeted S100A4 inhibition also modulated inflammation- and fibrosis-relevant gene sets in precision cut SSc skin slices in an ex vivo trial approach. Selected downstream targets of S100A4, such as AMP-activated protein kinase, calsequestrin-1, and phosphorylated STAT3, were validated on the protein level, and STAT3 inhibition was shown to prevent the profibrotic effects of S100A4 on fibroblasts in human skin. CONCLUSION: Inhibition of S100A4 confers dual targeting of inflammatory and fibrotic pathways in complementary mouse models of fibrosis and in SSc skin. These effects support the further development of anti-S100A4 mAbs as disease-modifying targeted therapies for SSc.

Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis.

OBJECTIVES: We have studied the damage-associated molecular pattern protein S100A4 as a driver of fibroblast activation in systemic sclerosis (SSc). METHODS: S100A4 protein concentration was measured by ELISA in serum of SSc (n=94) and healthy controls (n=15). Protein expression in skin fibroblast cultures from diffuse cutaneous SSc (SScF, n=6) and healthy controls (normal fibroblasts (NF), n=6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were tested on SScF and NF. RESULTS: Median (range) S100A4 (ng/mL) was higher in serum of SSc (89.9 (15.0-240.0)) than healthy controls (71.4 (7.9-131.8); p=0.027). There was association with SSc-interstitial lung disease (p=0.025, n=55), scleroderma renal crisis (p=0.026, n=4). Median (range) S100A4 (ng/mL) was higher in culture supernatants of SScF (4.19 (0.52-8.42)) than NF controls (0.28 (0.02-3.29); p<0.0001). AX-202 reduced the constitutive profibrotic gene and protein expression phenotype of SScF. Genome-wide RNA sequencing analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (false discovery rate (FDR) <0.001 and fold change (FC) >1.5) induced in NF by S100A4 were also constitutively overexpressed, and downregulated by AX-202, in SScF. Pathway mapping of these S100A4 dependent genes in SSc showed the most significant enriched Kegg pathways (FDR <0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). CONCLUSION: Our findings provide compelling evidence for a profibrotic role for S100A4 in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. This study supports examining the therapeutic potential of targeting S100A4 in SSc.

Neutralization of S100A4 induces stabilization of atherosclerotic plaques: role of smooth muscle cells.

AIMS: During atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they switch from a contractile to a synthetic phenotype. From porcine coronary artery, we isolated spindle-shaped (S) SMCs exhibiting features of the contractile phenotype and rhomboid (R) SMCs typical of the synthetic phenotype. S100A4 was identified as a marker of R-SMCs in vitro and intimal SMCs, in pig and man. S100A4 exhibits intra- and extracellular functions. In this study, we investigated the role of extracellular S100A4 in SMC phenotypic transition. METHODS AND RESULTS: S-SMCs were treated with oligomeric recombinant S100A4 (oS100A4), which induced nuclear factor (NF)-κB activation. Treatment of S-SMCs with oS100A4 in combination with platelet-derived growth factor (PDGF)-BB induced a complete SMC transition towards a pro-inflammatory R-phenotype associated with NF-κB activation, through toll-like receptor-4. RNA sequencing of cells treated with oS100A4/PDGF-BB revealed a strong up-regulation of pro-inflammatory genes and enrichment of transcription factor binding sites essential for SMC phenotypic transition. In a mouse model of established atherosclerosis, neutralization of extracellular S100A4 decreased area of atherosclerotic lesions, necrotic core, and CD68 expression and increased α-smooth muscle actin and smooth muscle myosin heavy chain expression. CONCLUSION: We suggest that the neutralization of extracellular S100A4 promotes the stabilization of atherosclerotic plaques. Extracellular S100A4 could be a new target to influence the evolution of atherosclerotic plaques.

Anti-S100A4 Antibody Therapy Is Efficient in Treating Aggressive Prostate Cancer and Reversing Immunosuppression: Serum and Biopsy S100A4 as a Clinical Predictor.

S100A4 oncoprotein plays a critical role during prostate cancer progression and induces immunosuppression in host tissues. We hypothesized that S100A4-regulated oncogenic activity in immunosuppressed prostate tumors promotes growth of neoplastic cells, which are likely to become aggressive. In the current study, we investigated whether biopsy-S100A4 gene alteration independently predicts the outcome of disease in patients and circulatory-S100A4 is druggable target for treating immunosuppressive prostate cancer. Aided by DECIPHER-genomic test, we show biopsy-S100A4 overexpression as predictive of (i) poor ADT response and (ii) high risk of mortality in 228 radical prostatectomy-treated patients. Furthermore, analysis of tumor genome data of more than 1,000 patients with prostate cancer (PRAD/SU2C/FHCRC studies) validated the association of S100A4-alteration to poor survival and metastasis. We show that increased serum-S100A4 levels are associated to the prostate cancer progression in patients. The prerequisite for metastasis is the escape of tumor cells via vascular system. We show that extracellular-S100A4 protein as a growth factor induces vascular transmigration of prostate cancer cells and bone demineralization thus forms an ideal target for therapies for treating prostate cancer. By employing surface plasmon resonance and isothermal titration calorimetry, we show that mab6B12 antibody interacts with and neutralizes S100A4 protein. When tested for therapeutic efficacy, the mab6B12 therapy reduced the (i) osteoblastic demineralization of bone-derived MSCs, (ii) S100A4-target (NFκB/MMP9/VEGF) levels in prostate cancer cells, and (iii) tumor growth in a TRAMPC2 syngeneic mouse model. The immuno-profile analysis showed that mAb6B12-therapy (i) shifted Th1/Th2 balance (increased Stat4+/T-bet+ and decreased GATA2+/CD68+/CD45+/CD206+ cells); (ii) modulated cytokine levels in CD4+ T cells; and (iii) decreased levels of IL5/6/12/13, sTNFR1, and serum-RANTES. We suggest that S100A4-antibody therapy has clinical applicability in treating immunosuppressive prostate cancer in patients.

Neurotrophic Effects of Vascular Endothelial Growth Factor B and Novel Mimetic Peptides on Neurons from the Central Nervous System.

Vascular endothelial growth factor B (VEGFB) is a pleiotropic trophic factor, which in contrast to the closely related VEGFA is known to have a limited effect on angiogenesis. VEGFB improves survival in various tissues including the nervous system, where the effect was observed mainly for peripheral neurons. The neurotrophic effect of VEGFB on central nervous system neurons has been less investigated. Here we demonstrated that VEGFB promotes neurite outgrowth from primary cerebellar granule, hippocampal, and retinal neurons in vitro. VEGFB protected hippocampal and retinal neurons from both oxidative stress and glutamate-induced neuronal death. The VEGF receptor 1 (VEGFR1) is required for VEGFB-induced neurotrophic and neuroprotective effects. Using a structure-based approach, we designed short peptides, termed Vefin1-7, mimicking the binding interface of VEGFB to VEGFR1. Vefins were analyzed for their secondary structure and binding to VEGF receptors and compared with previously described peptides derived from VEGFA, another ligand of VEGFR1. We show that Vefins have neurotrophic and neuroprotective effects on primary hippocampal, cerebellar granule, and retinal neurons in vitro with potencies comparable to VEGFB. Similar to VEGFB, Vefins were not mitogenic for MCF-7 cancer cells. Furthermore, one of the peptides, Vefin7, even dose-dependently inhibited the proliferation of MCF-7 cells in vitro. Unraveling the neurotrophic and neuroprotective potentials of VEGFB, the only nonangiogenic factor of the VEGF family, is promising for the development of neuroprotective peptide-based therapies.

The Multifaceted S100A4 Protein in Cancer and Inflammation.

The metastasis-promoting S100A4 protein, a member of the S100 family, has recently been discovered as a potent factor implicated in various inflammation-associated diseases. S100A4 is involved in a range of biological functions such as angiogenesis, cell differentiation, apoptosis, motility, and invasion. Moreover, S100A4 is also a potent trigger of inflammatory processes and induces the release of cytokines and growth factors under different pathological conditions.Indeed, the release of S100A4 upon stress and mainly its pro-inflammatory role emerges as the most decisive activity in disease development, such as rheumatoid arthritis (RA), systemic sclerosis (SSc) allergy, psoriasis, and cancer. In the scope of this review, we will focus on the role of S100A4 as a mediator of pro-inflammatory pathways and its associated biological processes involved in the pathogenesis of various human noncommunicable diseases (NCDs) including cancer.

The S100A4 Protein Signals through the ErbB4 Receptor to Promote Neuronal Survival.

Understanding the mechanisms of neurodegeneration is crucial for development of therapies to treat neurological disorders. S100 proteins are extensively expressed in the injured brain but S100's role and signalling in neural cells remain elusive. We recently demonstrated that the S100A4 protein protects neurons in brain injury and designed S100A4-derived peptides mimicking its beneficial effects. Here we show that neuroprotection by S100A4 involves the growth factor family receptor ErbB4 and its ligand Neuregulin 1 (NRG), key regulators of neuronal plasticity and implicated in multiple brain pathologies. The neuroprotective effect of S100A4 depends on ErbB4 expression and the ErbB4 signalling partners ErbB2/Akt, and is reduced by functional blockade of NRG/ErbB4 in cell models of neurodegeneration. We also detect binding of S100A4 with ErbB1 (EGFR) and ErbB3. S100A4-derived peptides interact with, and signal through ErbB, are neuroprotective in primary and immortalized dopaminergic neurons, and do not affect cell proliferation/motility - features which make them promising as potential neuroprotectants. Our data suggest that the S100-ErbB axis may be an important mechanism regulating neuronal survival and plasticity.

S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance.

BACKGROUND: The tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating the metastatic spread of tumor cells is the S100A4 protein. S100A4 is known as an inducer of inflammatory processes and has been shown to attract T-cells to the primary tumor and to the pre-metastatic niche. The present study aims to examine the immunomodulatory role of S100A4 in vivo and in vitro and assess the mode of action of 6B12, a S100A4 neutralizing antibody. METHODS: The therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche formation we determined the expression of metastatic niche markers. The levels of cytokine expression were assessed using antibody as well as PCR arrays and the results confirmed by qRT-PCR and ELISA. T-cell phenotyping and in vitro differentiation analyses were performed by flow cytometry. RESULTS: We show that the S100A4 protein alters the expression of transcription factor and signal transduction pathway genes involved in the T-cell lineage differentiation. T-cells challenged with S100A4 demonstrated reduced proportion of Th1-polarized cells shifting the Th1/Th2 balance towards the Th2 pro-tumorigenic phenotype. The 6B12 antibody restored the Th1/Th2 balance. Furthermore, we provide evidence that the 6B12 antibody deploys its anti-metastatic effect, by suppressing the attraction of T-cells to the site of primary tumor and pre-metastatic niche. This was associated with delayed primary tumor growth, decreased vessel density and inhibition of metastases. CONCLUSION: The S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts as an immunomodulatory agent and thus supports the view that the 6B12 antibody is a promising therapeutic candidate to fight cancer.

A generally applicable translational strategy identifies S100A4 as a candidate gene in allergy.

The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.

In vitro validation of an ultra-sensitive scanning fluorescence microscope for analysis of circulating tumor cells.

Analysis of circulating tumor cells (CTC) holds promise of providing liquid biopsies from patients with cancer. However, current methods include enrichment procedures. We present a method (CytoTrack), where CTC from 7.5 mL of blood is stained, analyzed and counted by a scanning fluorescence microscope. The method was validated by breast cancer cells (MCF-7) spiked in blood from healthy donors. The number of cells spiked in each blood sample was exactly determined by cell sorter and performed in three series of three samples spiked with 10, 33 or 100 cells in addition with three control samples for each series. The recovery rate of 10, 33 and 100 tumor cells in a blood sample was 55%, 70% and 78%, percent coefficient of variation (CV%) for samples was 59%, 32% and 18%, respectively. None of the control samples contained CTC. In conclusion, the method has been validated to highly sensitively detect breast cancer cells in spiking experiments and should be tested on blood samples from breast cancer patients. The method could benefit from automation that could reduce the CV%, and further optimization of the procedure to increase the recovery.

Peptide mimetic of the S100A4 protein modulates peripheral nerve regeneration and attenuates the progression of neuropathy in myelin protein P0 null mice.

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.

Anti-S100A4 antibody suppresses metastasis formation by blocking stroma cell invasion.

The small Ca-binding protein, S100A4, has a well-established metastasis-promoting activity. Moreover, its expression is tightly correlated with poor prognosis in patients with numerous types of cancer. Mechanistically, the extracellular S100A4 drives metastasis by affecting the tumor microenvironment, making it an attractive target for anti-cancer therapy. In this study, we produced a function-blocking anti-S100A4 monoclonal antibody with metastasis-suppressing activity. Antibody treatment significantly reduced metastatic burden in the lungs of experimental animals by blocking the recruitment of T cells to the site of the primary tumor. In vitro studies demonstrated that this antibody efficiently reduced the invasion of T cells in a fibroblast monolayer. Moreover, it was capable of suppressing the invasive growth of human and mouse fibroblasts. We presume therefore that the antibody exerts its activity by suppressing stroma cell recruitment to the site of the growing tumor. Our epitope mapping studies suggested that the antibody recognition site overlaps with the target binding interface of human S100A4. We conclude here that this antibody could serve as a solid basis for development of an efficient anti-metastatic therapy.

α-Catenin contributes to the strength of E-cadherin-p120 interactions.

Cadherin-catenin interactions play an important role in cadherin-mediated adhesion. Here we present strong evidence that in the cadherin-catenin complex α-catenin contributes to the binding strength of another catenin, p120, to the same complex. Specifically, we found that a ß-catenin-uncoupled cadherin mutant interacts much more weakly with p120 than its full-size counterpart and that it is rapidly endocytosed from the surface of A-431 cells. We also showed that p120 overexpression stabilizes this mutant on the cell surface. Examination of the α-catenin-deficient MDA-MB-468 cells and their derivates in which α-catenin was reintroduced showed that α-catenin reinforces E-cadherin-p120 association. Finally, a cross-linking analysis of the cadherin-catenin complex indicated that a large loop located in the middle of the p120 arm-repeat domain is in close spatial vicinity to the amino-terminal VH1 domain of α-catenin. The six amino acid-long extension of this loop, caused by an alternative splicing, weakens p120 binding to cadherin. The data suggest that α-catenin-p120 contact within the cadherin-catenin complex can regulate cadherin trafficking.

Role of fibulin-5 in metastatic organ colonization.

The tumor microenvironment is now recognized as a major factor in determining the survival and growth of disseminated tumor cells at potential metastatic sites. Tumor cells send signals to stroma cells and stimulate them to produce factors that in turn create favorable conditions for tumor cell metastasis. Activated fibroblasts constitute an important component of the tumor-associated stroma. We have previously shown that S100A4 protein produced by stromal fibroblasts in the primary tumor stimulates metastasis formation. Here we show that activated fibroblasts also stimulate the formation of metastases independently of S100A4 expression during organ colonization. To identify genes that could potentially interfere with fibroblast-driven metastasis, we used gene expression profiling of S100A4-deficient fibroblasts treated with and without tumor cell-conditioned media. Five differentially expressed genes encoding cell surface and secreted proteins with potential metastasis-modulating activity were selected. Expression of lymphocyte antigen 6 complex (Ly6c) and matrix metalloproteinase 3 (Mmp3) was upregulated in fibroblasts in response to tumor-conditioned medium, whereas expression of cadherin-16 (Cdh16), Ccn2, and fibulin-5 (Fbln5) was downregulated. Further analysis showed that Fibulin-5 is able to suppress the metastatic colonization of lungs and liver. Additional studies suggest a mechanism in which Fibulin-5 suppresses metastasis formation by inhibiting production of matrix metalloproteinase 9 (MMP9) and reducing the invasive behavior of fibroblasts. Together our data are consistent with the notion that tumors secrete factors that downregulate expression of Fbln5 in fibroblasts at sites of metastatic colonization, in turn upregulating Mmp9 expression and stimulating metastatic organ colonization.

Metastasis-inducing S100A4 and RANTES cooperate in promoting tumor progression in mice.

BACKGROUND: The tumor microenvironment has been described as a critical milieu determining tumor growth and metastases. A pivotal role of metastasis-inducing S100A4 in the development of tumor stroma has been proven in animal models and verified in human breast cancer biopsies. Expression and release of S100A4 has been shown in various types of stroma composing cells, including fibroblasts and immune cells. However, the events implicated in upstream and downstream pathways regulating the activity of the extracellular S100A4 protein in the tumor milieu remain unsolved. METHODOLOGY/PRINCIPAL FINDINGS: We studied the interplay between the tumor cell-derived cytokine regulated-upon-activation, normal T-cell expressed and secreted (RANTES; CCL5) and S100A4 which were shown to be critical factors in tumor progression. We found that RANTES stimulates the externalization of S100A4 via microparticle shedding from the plasma membrane of tumor and stroma cells. Conversely, the released S100A4 protein induces the upregulation of fibronectin (FN) in fibroblasts and a number of cytokines, including RANTES in tumor cells as well as stimulates cell motility in a wound healing assay. Importantly, using wild type and S100A4-deficient mouse models, we demonstrated a substantial influence of tumor cell-derived RANTES on S100A4 release into blood circulation which ultimately increases the metastatic burden in mice. CONCLUSIONS/SIGNIFICANCE: Altogether, the data presented strongly validate the pro-metastatic function of S100A4 in the tumor microenvironment and define how the tumor cell-derived cytokine RANTES acts as a critical regulator of S100A4-dependent tumor cell dissemination. Additionally, for the first time we demonstrated the mechanism of S100A4 release associated with plasma membrane microparticle shedding from various cells types.

Lung metastasis fails in MMTV-PyMT oncomice lacking S100A4 due to a T-cell deficiency in primary tumors.

Interactions between tumor and stroma cells are essential for the progression of cancer from its initial growth at a primary site to its metastasis to distant organs. The metastasis-stimulating protein S100A4 exerts its function as a stroma cell-derived factor. Genetic depletion of S100A4 significantly reduced the metastatic burden in lungs of PyMT-induced mammary tumors. In S100A4(+/+) PyMT mice, massive leukocyte infiltration at the site of the growing tumor at the stage of malignant transition was associated with increased concentration of extracellular S100A4 in the tumor microenvironment. In contrast, in S100A4(-/-) PyMT tumors, a significant suppression of T-cell infiltration was documented at the transition period. In vitro, the S100A4 protein mediated the attraction of T cells. Moreover, S100A4(+/+), but not S100A4(-/-), fibroblasts stimulated the invasion of T lymphocytes into fibroblast monolayers. In vivo, the presence of S100A4(+/+), but not S100A4(-/-), fibroblasts significantly stimulated the attraction of T lymphocytes to the site of the growing tumor. Increased levels of T cells were also observed in the premetastatic lungs of tumor-bearing mice primed to metastasize by S100A4(+/+) fibroblasts. Treatment of T cells with the S100A4 protein stimulated production of cytokines, particularly granulocyte colony-stimulating factor and eotaxin-2. The same cytokines were detected in the fluid of S100A4(+/+) PyMT tumors at the transition period. We suggest that release of S100A4 in the primary tumor stimulates infiltration of T cells and activates secretion of cytokines, thus triggering sequential events that fuel tumor cells to metastasize. Similar processes could occur in the premetastatic lungs, facilitating generation of inflammatory milieu favorable for metastasis formation.

Metastasis-inducing S100A4 protein is associated with the disease activity of rheumatoid arthritis.

OBJECTIVES: To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients. METHODS: Plasma levels of the S100A4 protein were analysed in 40 anti-TNF-alpha naive patients with active RA. Of the 40 patients, 25 were treated with adalimumab and monitored over time. The conformational form of S100A4 was analysed using size-exclusion gel chromatography. TNF-alpha mRNA expression and protein synthesis were analysed by RT-PCR and ELISA, respectively. RESULTS: Baseline levels of S100A4 were significantly correlated with disease activity in RA patients (r = 0.41; P < 0.01). After 12 weeks of treatment with adalimumab, there was an obvious shift in the conformations of S100A4 from the multimeric to the dimeric forms, whereas the total levels of the S100A4 protein remained unchanged. This suggests that the bioactive (multimer) S100A4 may decline in response to successful treatment with adalimumab. In addition, we showed significant up-regulation of TNF-alpha mRNA (P < 0.01), and protein release to the cell culture medium of monocytes stimulated with the S100A4 multimer compared with those treated with the dimer and to the unstimulated monocytes (P < 0.001). CONCLUSIONS: This is the first study to show that the levels of the S100A4 protein are correlated with RA disease activity. Furthermore, only the bioactive form, but not the total amount of S100A4, decreases after successful TNF-alpha blocking therapy in patients with RA. These data support an important role for the S100A4 multimer in the pathogenesis of RA.

Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein.

The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin-mediated proliferation of mouse embryonic fibroblasts. S100A4-neutralizing antibodies, as well as EGFR- and ErbB2 receptor-specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation. Structured digital abstract: * MINT-7256556: EGF (uniprotkb:P01133) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256512: BC (uniprotkb:P35070) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256485, MINT-7256618, MINT-7256636: AR (uniprotkb:P15514) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256494: HB-EGF (uniprotkb:Q99075) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256502: P53 (uniprotkb:P04637) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256654: S100A2 (uniprotkb:P29034) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256693: S100A5 (uniprotkb:P33763) binds (MI:0407) to AR (uniprotkb:P15514) by far western blotting (MI:0047) * MINT-7256593: S100A4 (uniprotkb:P26447) binds (MI:0407) to BC (uniprotkb:P35070) by pull down (MI:0096) * MINT-7256567: S100A4 (uniprotkb:P26447) binds (MI:0407) to AR (uniprotkb:P15514) by pull down (MI:0096).