The involvement of Mts1(S100A4), a small Ca 2+ -binding protein in tumor progression and metastasis had been demonstrated. However, the mechanism by which mts1(S100A4) promoted metastasis had not been identi®ed. Here we demonstrated that Mts1(S100A4) had signi®cant stimulatory eect on the angiogenesis. We detected high incidence of hemangiomas ± benign tumors of vascular origin in aged transgenic mice ubiquitously expressing the mts1(S100A4) gene. Furthermore, the serum level of the Mts1(S100A4) protein increased with ageing. Tumors developed in Mts1-transgenic mice revealed an enhanced vascular density. We showed that an oligomeric, but not a dimeric form of the Mts1(S100A4) protein was capable of enhancing the endothelial cell motility in vitro and stimulate the corneal neovascularization in vivo. An oligomeric fraction of the protein was detected in the conditioned media as well as in human serum. The data obtained allowed us to conclude that mts1(S100A4) might induce tumor progression via stimulation of angiogenesis. Oncogene (2001) 20, 4685 ± 4695.

S100A4(mts1) protein expression has been strongly associated with metastatic tumor progression. It has been suggested as a prognostic marker for a number of human cancers. It is proposed that extracellular S100A4 accelerates cancer progression by stimulating the motility of endothelial cells, thereby promoting angiogenesis. Here we show that in 3D culture mouse endothelial cells (SVEC 4-10) respond to recombinant S100A4 by stimulating invasive growth of capillary-like structures. The outgrowth is not dependent on the stimulation of cell proliferation, but rather correlates with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. b-Casein zymography demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis-stimulating activity of S100A4(mts1).

The S100A4(mts1) protein stimulates metastatic spread of tumor cells. An elevated expression of S100A4 is associated with poor prognosis in many human cancers. Dynamics of tumor development were studied in S100A4-deficient mice using grafts of CSML100, highly metastatic mouse mammary carcinoma cells. A significant delay in tumor uptake and decreased tumor incidences were observed in S100A4(À/À) mice compared with the wild-type controls. Moreover, tumors developed in S100A4(À/À) mice never metastasize. Immunohistochemical analyses of these tumors revealed reduced vascularity and abnormal distribution of host-derived stroma cells. Coinjection of CSML100 cells with immortalized S100A4(+/+) fibroblasts partially restored the dynamics of tumor development and the ability to form metastasis. These fibroblasts were characterized by an enhanced motility and invasiveness in comparison with S100A4(À/À) fibroblasts, as well as by the ability to release S100A4 into the tumor environment. Taken together, our results point to a determinative role of host-derived stroma cells expressing S100A4 in tumor progression and metastasis. (Cancer Res 2005; 65(9): 3772-80)

Causal implication of S100A4 in inducing metastases was convincingly shown previously. However, the mechanisms that associate S100A4 with tumor progression are not well understood. S100A4 protein, as a typical member of the S100 family, exhibits dual, intracellular and extracellular, functions. This work is focused on the extracellular function of S100A4, in particular its involvement in tumor-stroma interplay in VMR (mouse adenocarcinoma cell line) tumor cells, which exhibit stroma-dependent metastatic phenotype. We demonstrated the reciprocal influence of tumor and stroma cells where tumor cells stimulate S100A4 secretion from fibroblasts in culture. In turn, extracellular S100A4 modifies the cytoskeleton and focal adhesions and triggers several other events in tumor cells. We found stabilization of the tumor suppressor protein p53 and modulation of its function. In particular, extracellular S100A4 down-regulates the pro-apoptotic bax and the angiogenesis inhibitor thrombospondin-1 genes. For the first time, we demonstrate here that the S100A4 protein added to the extracellular space strongly stimulates proteolytic activity of VMR cells. This activity most probably is associated with matrix metalloproteinases and, in particular, with matrix metalloproteinase-13. Finally, the application of the recombinant S100A4 protein confers stroma-independent metastatic phenotype on VMR tumor cells. In conclusion, our results indicate that metastasis-inducing S100A4 protein plays a pivotal role in the tumor-stroma environment. S100A4 released either by tumor or stroma cells triggers pro-metastatic cascades in tumor cells.Metastatic dissemination of cancer cells results in incurable disease and becomes one of the leading causes of cancer patient deaths. Studies on genes and their protein products involved in this process are of great importance.Causal implication of S100A4 (mts1, CAPL, pEL98, Calvasculin, p9Ka, and FSP1) in metastatic tumor progression was demonstrated by several approaches. Transfection of rodent and human non-metastatic tumor cell lines with S100A4 converts their phenotype to metastatic cells, and contrariwise, the antisense-and ribozyme-mediated mts1-inactivation abolish the metastatic potential of metastatic tumor cells (1-4). Tumors developed in transgenic mice bearing exogenous S100A4 gene acquired metastatic phenotype (5, 6). The tight association of S100A4 with metastasis allows us to rate it among the most reliable prognostic markers. A high level of S100A4 in various types of cancers (breast, esophageal, gastric, colorectal, bladder, gallbladder, and lung) correlates with unfavorable prognosis and lethality (7-11). S100A4 belongs to the S100 family of Ca 2ϩ -binding proteins that comprises 20 members. They are involved in the regulation of various important cellular functions such as cell growth, cell-cell communication, energy metabolism, contraction, neurite outgrowth, and cell motility (for review see Refs. 12-14). S100A4 protein, as a typical member of the S100 family, exerts dual, intracellul...

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra-and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca 2؉ signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca 2؉ entry via nonselective cation channels and via T-and L-type voltage-gated Ca 2؉ channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders.The S100 family is a group of vertebrate-specific Ca 2ϩ -binding proteins with a highly conserved primary structure possessing both intra-and extracellular functions. Most S100 family members, including S100A4, are antiparallelly packed homodimers stabilized by disulfide bridges (reviewed in references 8 and 9). Intracellularly, S100 proteins are involved in a variety of processes, including the regulation of cytoskeletal dynamics, Ca 2ϩ homeostasis, and cell proliferation and differentiation. Importantly, some S100 proteins can also be secreted, form oligomers owing to the nonreducing conditions of the environment, and exert their effects acting at the cell surface (10; 43; reviewed in reference 20). A plasma membrane target for S100B and S100A12, the receptor for advanced glycation end products (RAGE), has been identified on inflammatory and neural cells (14). However, RAGE is probably not the sole receptor for members of the S100 family, since the effects of extracellular S100A12 and S100B proteins can be observed in cells lacking RAGE (32), and some of these effects are RAGE independent in cells expressing the receptor (37).The S100A4 (also termed Mts1) gene was isolated from tumor cells (11,40), where its expression increased the ability of the tumor to metastasize. S100A4 has also been detected in healthy tissues, particularly in the nervous system. In both the brain and spinal cord, S100A4 expression appears in astrocytes shortly after the start of myelination, with the highest level observed in the areas in which neurogenesis takes place and in regions possessing high plasticit...

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 upregulation 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.

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). ' 2007 Wiley-Liss, Inc.Key words: breast cancer; tumor stroma; S100A4The S100 protein family is the largest subgroup within the superfamily of proteins carrying the Ca 21 -binding EF-hand motif. Members of this family are multifunctional signaling proteins that are involved in the regulation of diverse cellular processes such as contraction, motility, cell growth, differentiation, cell cycle progression, transcription, and secretion. 1 Diseases such as cardiomyopathies, neurodegenerative and inflammatory disorders, as well as cancer are associated with altered S100 protein levels. 2 Despite their small molecular size and their conserved functional domain of 2 distinct EF-hands, these proteins exhibit a wide-range of tissue-specific intra and extracellular functions.Similar to the other members of this protein family, S100A4 exhibits diverse functions. 1 Several lines of evidence based either on spontaneous metastasis formation in rodent or experimental models utilizing human cancer cell lines have suggested a causative role for this protein in the metastatic process. 3,4 In particular, experiments with transgenic mouse models of breast cancer have shown that the expression levels of S100A4 correlates with metastasis. 5,6 Moreover, recent studies by Xue et al. have shown that transgenic mice expressing the Polyoma virus middle T antigen when crossed with mice carrying null alleles for S100A4 exhibit a significant decrease in lung metastases. 7 It has been hypothesized that S100A4 stimulates metastatic spread of tumor cells by stimulating motility via interaction with its target proteins, such as nonmuscle myosin. [8][9][10][11][12] S100A4 has also, from studies on renal diseases in mice, been identified as the fibroblast specific protein 1 (...