The role of S100a4 (Mts1) in Apc- and Smad4-driven tumour onset and progression.

INTRODUCTION: S100a4 is a calcium-binding protein belonging to the family of S100-proteins, highly expressed in different stromal cell types. S100A4 has been reported as a prognostic marker in colorectal cancer in association with tumour progression and metastasis. METHODS: In this study, we analysed the in vivo role of S100a4 in intestinal tumour initiation and progression using different transgenic and knockout mouse models. RESULTS: We found that genetic ablation or overexpression of S100a4 in both Apc- and Smad4-mutant mice do not affect tumour initiation in the intestinal tract. In contrast, S100a4 epithelial overexpression in Apc1638N/+/KRASV12G mice increases the dissemination of intestinal tumour cells to the liver, in agreement with its role in tumour metastasis. Moreover, we report a novel role for S100a4 in desmoid formation where S100a4 deficiency results in a significant reduction of the tumour burden characteristic of the Apc1638N model. In agreement with these results, S100a4 appears to be co-expressed together with mesenchymal stem cell (MSC) markers in desmoid tumours from Apc1638N/+ mice, as well as from sporadic and hereditary human desmoids. CONCLUSION: Our data provide the first report on the in vivo role of S100a4 in intestinal tumourigenesis and describe a new role for S100a4 in the aetiology of desmoids formation.

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.

The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis.

OBJECTIVES: S100A4 has been implicated in cancer and several inflammatory diseases, including RA. The aim of the present study was to determine whether S100A4 can stimulate proinflammatory cytokine production in mononuclear cells. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from patients with RA were stimulated with S100A4, S100A8, S100A9 and S100A12. The production of IL-1ß, IL-6 and TNF-α was measured by ELISA. Receptor for advanced glycation end products (RAGEs) and Toll-like receptor 4 (TLR4) signalling were examined. For signalling pathway blocking studies, inhibitors of myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa B (NF-κB) and the mitogen activated protein (MAP) kinases p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) were used. MAP kinase activation was evaluated by western blotting. RESULTS: Stimulation of PBMCs with S100A4 significantly up-regulated IL-1ß, IL-6 and TNF-α production compared with unstimulated cells (P < 0.001). Importantly, the production of these cytokines was markedly enhanced in response to S100A4 compared with S100A8 and S100A12; however, it was less pronounced compared with S100A9. Furthermore, enhanced production of proinflammatory cytokines in S100A4-stimulated PMBCs was at least partly mediated via TLR4, but not RAGEs, and by activation of the transcription factor NF-κB and the MAP kinases p38 and ERK1/2. CONCLUSION: This is the first study to demonstrate that S100A4 can induce an inflammatory response mediated by TLR4 and by the activation of NF-κB and the kinases p38 and ERK1/2 in mononuclear cells from patients with RA. Therefore S100A4 may be a potential therapeutic target for immune-mediated diseases.

Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts.

Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown of S100A4 significantly suppressed both cell proliferation and collagen expressions. S100A4 co-localized and interacted with p53 in the nucleus. S100A4 knockdown increased the expression of p53-downstream genes, p21 and mdm2, and concomitant knockdown of p53 recovered cell proliferation and collagen expression. Transverse aortic constriction (TAC) was performed in S100A4 knockout (KO) mice, which showed a similar baseline-phenotype to wild type (WT) mice. Although there was no difference in hypertrophic response, KO mice showed reduced interstitial fibrosis, decreased myofibroblasts, and suppressed expressions of collagens and profibrotic cytokines in the left ventricle. Also, DNA microarray analysis showed that S100A4 knockout in vivo had a significant impact on expressions of p53-associated genes. These findings suggest that S100A4 modulates p53 function in fibroblasts and thereby mediates myocardial interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis.

The metastasis-promoting S100A4 protein confers neuroprotection in brain injury.

Identification of novel pro-survival factors in the brain is paramount for developing neuroprotective therapies. The multifunctional S100 family proteins have important roles in many human diseases and are also upregulated by brain injury. However, S100 functions in the nervous system remain unclear. Here we show that the S100A4 protein, mostly studied in cancer, is overexpressed in the damaged human and rodent brain and released from stressed astrocytes. Genetic deletion of S100A4 exacerbates neuronal loss after brain trauma or excitotoxicity, increasing oxidative cell damage and downregulating the neuroprotective protein metallothionein I+II. We identify two neurotrophic motifs in S100A4 and show that these motifs are neuroprotective in animal models of brain trauma. Finally, we find that S100A4 rescues neurons via the Janus kinase/STAT pathway and, partially, the interleukin-10 receptor. Our data introduce S100A4 as a therapeutic target in neurodegeneration, and raise the entire S100 family as a potentially important factor in central nervous system injury.

Building the niche: the role of the S100 proteins in metastatic growth.

Communication between cancer cells and stromal cells, often mediated by extracellular molecules in the tumor microenvironment, plays a central role in tumorigenesis and metastasis. The establishment of a pro-inflammatory milieu is increasingly recognized as an important consequence of these interactions. The family of S100 Ca2+-binding proteins has been implicated in many aspects of the interaction between cancer cells and stromal cells, and contributes to the formation of an inflammatory tumor microenvironment. Focusing on S100A4, S100A8 and S100A9, in this review we discuss the role these proteins play in primary tumors and in the development of metastases, in particular during the formation of pre-metastatic niches.

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.

The metastasis promoting protein S100A4 is increased in idiopathic inflammatory myopathies.

OBJECTIVES: The S100A4 protein is known as a metastasis promoting factor; however, its involvement in non-malignant diseases such as RA and psoriasis has been recently described. The aim of this study was to investigate the expression and possible role of S100A4 in idiopathic inflammatory myopathies. METHODS: S100A4 protein expression was detected by immunohistochemistry in muscle tissue from control individuals (n = 11) and patients with PM and DM (n = 8/6). IF staining was used to co-localize S100A4 with selected cells. Cytokine expression and protein synthesis in S100A4-treated cells were analysed by RT-PCR and ELISA. RESULTS: S100A4 protein was significantly up-regulated in muscle tissue of patients with inflammatory myopathies compared with control individuals and was associated particularly with the presence of mononuclear infiltrates. Only few regenerating muscle fibres in PM/DM expressed S100A4. Then we analysed the effect of S100A4 on human myocytes and peripheral blood mononuclear cells (PBMCs). Although S100A4 did not affect myocytes, stimulation of PBMCs with S100A4 significantly induced the expression and synthesis of TNF-α, IL-1ß and IL-6, but not of IFN-α. We showed that S100A4 is not directly involved in perforin/granzyme B-induced apoptosis and that it does not modulate the expression of Bax and Bcl2 mRNA in myocytes and PBMCs. CONCLUSION: Increased expression of S100A4 in inflamed muscle tissue highlights its potential role in the pathogenesis of inflammatory myopathies. S100A4 may act as a cytokine-like factor indirectly promoting muscle fibre damage by stimulating mononuclear cells to increase the synthesis of pro-inflammatory cytokines.

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.

Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts.

INTRODUCTION: Tumour phenotype is regulated in a complex fashion as a result of interactions between malignant cells and the tumour stroma. Fibroblasts are the most abundant and perhaps most active part of the tumour stroma. A better understanding of the changes that occur in fibroblasts in response to the presence of malignant cells may lead to the development of new strategies for cancer treatment. We explored the effects of fibroblasts on the growth and invasion of mammary carcinoma tumour cells in vitro and in vivo. METHODS: In order to analyse secreted factors that affect invasive abilities of breast cancer cells we co-cultured human mammary fibroblasts (HMF3s) and cancer cells (MCF7S1) in three-dimensional (3D) growth conditions devoid of heterogeneous cell-cell contact. To study the possible influence of fibroblasts on MCF7S1 cancer cell growth in vivo we co-injected HMF3s and MCF7S1 cells in Balb/c nu/nu mice. RESULTS: In 3D co-culture both HMF3s and MCF7S1 cells demonstrated enhanced invasion into a Matrigel matrix. This was correlated with enhanced expression of the metastasis promoting S100A4 protein in fibroblasts, stimulation of the matrix metalloproteinase (MMP)-2 activity, and enhanced secretion of a range of different cytokines. Orthotopic injection of oestrogen-dependent MCF7S1 cancer cells together with fibroblasts showed stimulation of tumour growth in mice without an external oestrogen supply. The resulting tumours were characterized by increased development of extracellular matrix, as well as an increase of murine S100A4 concentration and activity of MMP-2 in the tumour interstitial fluid. CONCLUSION: Stimulation of the invasive phenotype of tumour cells in 3D co-cultures with fibroblasts could be correlated with increased production of S100A4 and MMP-2. We propose that enhanced development of mouse host-derived tumour stroma in a MCF7S1 co-injection xenograft model leads to oestrogen independency and is triggered by the initial presence of human fibroblasts.

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.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

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).

Immunogenicity of HLA-A1-restricted peptides derived from S100A4 (metastasin 1) in melanoma patients.

S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.

Metastasis-inducing S100A4 protein: implication in non-malignant human pathologies.

The role of S100A4 in tumor progression and metastasis is well documented in numerous research articles and summarized in several reviews. Currently S100A4 is categorized as an essential metastasis-promoting factor whose production and secretion from "activated" stromal cells (fibroblasts, immunocytes and vascular cells) is initiated and stimulated by signals derived in tumor cells (cytokines, growth factors and others). However recent data gained from experimental and clinical studies significantly extend our knowledge on S100A4. Implications of S100A4 in various non-malignant pathological conditions have been demonstrated by number of research groups. In the mini-review we attempted to highlight the role of S100A4 in other than cancer important human pathologies, such as autoimmune inflammation (RA) and disorders in cardio-vascular, nervous and pulmonary systems. We suggest that diverse human diseases might have common molecular components and pathway(s). Possibly, inflammatory machinery and S100A4 as its intrinsic constituent could contribute to the pathogenesis of various disorders. Therefore, we presume that facts on S100A4 performance could be attractive for broad range of researchers and clinicians.

Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer.

The S100A4 protein, which is involved in the metastasis process, is a member of the S100 superfamily of Ca-binding proteins. Members of this family are multifunctional signaling proteins with dual extra and intracellular functions involved in the regulation of diverse cellular processes. Several studies have established a correlation between S100A4 protein expression and worse prognosis for patients with various malignancies including breast cancer. In this article, we have used specific antibodies in combination with immunohistochemistry (IHC) to identify the cell types that express S100A4 in human breast cancer biopsies obtained from high-risk patients. IHC analysis of 68 tumor biopsies showed that the protein is expressed preferentially by various cell types present in the tumor microenvironment (macrophages, fibroblasts, activated lymphocytes), rather than by the tumor cells themselves. Moreover, we show that the protein is externalized by the stroma cells to the fluid that bathes the tumor microenvironment, where it is found in several forms that most likely correspond to charge variants. Using a specific ELISA test, we detected a significant higher concentration of S100A4 in the tumor interstitial fluid (TIF) as compared to their corresponding normal counterparts (NIF).

S100A4 is upregulated in injured myocardium and promotes growth and survival of cardiac myocytes.

OBJECTIVE: The multifunctional Ca2+-binding protein S100A4 (also known as Mts1 and Fsp1) is involved in fibrosis and tissue remodeling in several diseases including cancer, kidney fibrosis, central nervous system injury, and pulmonary vascular disease. We previously reported that S100A4 mRNA expression was increased in hypertrophic rat hearts and that it has pro-cardiomyogenic effects in embryonic stem cell-derived embryoid bodies. We therefore hypothesized that S100A4 could play a supportive role in the injured heart. METHODS AND RESULTS: Here we verify by quantitative real-time PCR and immunoblotting that S100A4 mRNA and protein is upregulated in hypertrophic rat and human hearts and show by way of confocal microscopy that S100A4 protein, but not mRNA, appears in cardiac myocytes only in the border zone after an acute ischemic event in rat and human hearts. In normal rat and human hearts, S100A4 expression primarily colocalizes with markers of fibroblasts. In hypertrophy elicited by aortic banding/stenosis or myocardial infarction, this expression is increased. Moreover, invading macrophages and leucocytes stain strongly for S100A4, further increasing cardiac levels of S100A4 protein after injury. Promisingly, recombinant S100A4 protein elicited a robust hypertrophic response and increased the number of viable cells in cardiac myocyte cultures by inhibiting apoptosis. We also found that ERK1/2 activation was necessary for both the hypertrophy and survival effects of S100A4 in vitro. CONCLUSIONS: Along with proposed angiogenic and cell motility stimulating effects of S100A4, these findings suggest that S100A4 can act as a novel cardiac growth and survival factor and may have regenerative effects in injured myocardium.

Up-regulation of metastasis-promoting S100A4 (Mts-1) in rheumatoid arthritis: putative involvement in the pathogenesis of rheumatoid arthritis.

OBJECTIVE: To examine the involvement of the metastasis-inducing protein S100A4 (Mts-1) in the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue, synovial fluid, and plasma were obtained from RA and osteoarthritis (OA) patients who were undergoing joint surgery. Immunohistochemical and immunofluorescence analyses and enzyme-linked immunosorbent assays were used to determine the locations and concentrations of S100A4. The conformational structure of S100A4 in plasma and synovial fluid was determined after fractionation by size-exclusion chromatography, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis. Expression of various S100 proteins in RA synovium was determined by immunofluorescence and double-staining using specific anti-S100 antibodies. RESULTS: We found an up-regulation of S100A4 in cells infiltrating RA synovial tissue. Most cell types identified by cell-specific markers (fibroblasts, immune cells, and vascular cells) contributed to the production of S100A4 in RA synovial tissue. The pattern of S100A4 expression differed significantly from that of the proinflammatory proteins S100A9 and S100A12, which were restricted to phagocytes and granulocytes. The up-regulation of S100A4 in RA synovial tissue was consistent with the high concentrations of the protein in RA versus OA plasma (mean 1,100 versus 211 ng/ml) and synovial fluid (mean 1,980 versus 247 ng/ml). Moreover, we found that S100A4 in RA plasma and synovial fluid was present in bioactive multimeric (M-S100A4) conformations, whereas in OA, the majority of extracellular S100A4 was detected as the less active dimeric form. Consistent with our observations in tumor models, extracellular S100A4 stabilized the p53 tumor suppressor in RA synovial fibroblast-like cells and affected the regulation of p53 target genes, including Bcl-2, p21(WAF), and Hdm-2, as well as matrix metalloproteinases. CONCLUSION: Overexpression of S100A4 in RA synovial tissue and its release as M-S100A4 can influence p53 function and modulate the expression of several genes that are potentially implicated in the disease process. Thus, S100A4 might play an important role in the pathogenesis of RA and might represent a new target for the treatment of RA.

Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition.

Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.