S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

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.     

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.     

Extracellular S100A4 as a key player in fibrotic diseases

Fibrosis is characterized by fibroblast activation, extracellular matrix (ECM) accumulation and infiltration of inflammatory cells that sometimes leads to irreversible organ dysfunction. Considerable evidence now indicates that inflammation plays a critical role in the initiation and progression of organ fibrosis. S100A4 protein, a ubiquitous member of the S100 family, has recently been discovered as a potential factor implicated in fibrotic diseases. S100A4 protein is released at inflammatory site and has a certain biological function to promote cell motility, invasion, ECM remodelling, autophagy and angiogenesis. In addition, extracellular S100A4 is also a potential causation of inflammatory processes and induces the release of cytokines and growth factors under different pathological conditions. Elevated S100A4 level in patients’ serum closely correlates with disease activity in several fibrotic diseases and serves as a useful biomarker for diagnosis and monitoring disease progression. Analyses of knockout mouse models have identified a functional role of extracellular S100A4 protein in fibrotic diseases, suggesting that suppressing its expression, release or function might be a promising therapeutic strategy. This review will focus on the role of extracellular S100A4 as a key regulator of pro-inflammatory signalling pathways and its relative biological processes involved in the pathogenesis of fibrosis.     

S100A4与肿瘤细胞增殖及凋亡的研究进展

转移相关蛋白S100A4属于S100钙离子结合蛋白超家族,有着共同的EF手型功能区来介导其活性。S100A4在众多肿瘤生物学行为中起着调节作用。而且,S100A4在不同类型的肿瘤患者扮演着判断预后的角色。目前研究认为其与肿瘤细胞运动、增殖、刺激血管生成及基质重建有关系。本文就S100A4生物学特性及其对肿瘤细胞增殖、凋亡中的作用和可能机制作一综述。     

Evaluation of potential interactions between the metastasis-associated protein S100A4 and the tumor suppressor protein p53

Metastasis is a complex cascade of events involving a finely tuned interplay between malignant cells and multiple host factors. The transition from benign tumor growth to malignancy is manifested by the ability of tumor cells to traverse tissue barriers and invade surrounding tissues. Among a multitude of factors playing a role, the small calcium-binding protein S100A4 has been found to add to the invasive and metastatic capacity of cancer cells. However, the exact molecular function or mechanism by which S100A4 exerts its putative metastasis-promoting effects has not been fully elucidated, and the protein is most likely involved in several aspects of tumor progression. Several studies have recently described a direct interaction and/or reciprocal influence between S100A4 and the tumor suppressor protein p53. This corresponds to reports linking p53 to other S100-family members, especially S100B. The consequences are intriguing, connecting the metastasis-promoting protein S100A4 to the large set of important p53-mediated functions, with broad potential importance in cancer development and metastasis. In this review we emphasize the studies involving p53 and S100A4, elucidating and comparing reported results and conclusions.     

Matrix metalloproteinases in tumor-host cell communication

The microenvironment or stroma immediately surrounding tumor cells consists of a three-dimensional extracellular matrix (ECM) and stromal cells such as fibroblasts and inflammatory cells. The matrix metalloproteinases (MMPs) constitute a family of over 24 members, which collectively are capable of degrading virtually the entire ECM. Strict regulation of MMP expression is critical in order to maintain proper ECM homeostasis, but in disease states such as cancer there is often a high level of MMP activity at the tumor-stroma interface. Several studies have documented the importance of MMP-mediated ECM destruction in the successful dissemination of several tumor types, but it has become increasingly clear that they are also involved in earlier stages of tumorigenesis. MMPs are implicated in a wide variety of roles that can assist tumor initiation, growth, migration, angiogenesis, the selection of apoptosis-resistant subpopulations, and in invasion and metastasis. Interestingly, the factors responsible for many of these effects are derived from the cell surfaces of the tumor or stromal cells or are embedded in the ECM. Therefore, the MMPs can no longer be thought of solely as ECM destructionists, but as part of an elegant communication system through which the tumor interacts with the stroma.     

Metastatic potential of B16-F10 melanoma cells is enhanced by extracellular S100A4 derived from RAW264.7 macrophages

S100A4, synthesized and secreted from both tumor and stroma cells, modulates an aggressive tumor phenotype in various cancers by intracellular and extracellular interactions which are not completely understood. Because of the high content of tumor-associated macrophages in melanoma, here, a syngeneic model (coculture of mouse B16-F10 melanoma cells (Mel) and RAW264.7 macrophages (M?); administration (i.v.) of Mel and M?/Mel in NMRI nu/nu mice) was used to investigate synthesis and secretion of (a) S100A4, (b) S100A4-mediated signaling and activation of NFκB, and (c) S100A4-mediated modulation of Mel invasiveness in vitro (transwell assay, transwell matrigel assay) and in vivo (metastatic lung colonization), respectively. In this model substantial S100A4 synthesis and secretion is demonstrated in M?. Macrophage-derived S100A4 promotes Mel invasiveness in a paracrine manner in vitro, which is further substantiated in control experiments using recombinant human S100A4 and Mel stably transfected with mouse S100A4. Moreover, the participation of S100A4-mediated signaling, e.g., via the receptor for advanced glycation endproducts (RAGE), resulting in activation of NFκB was demonstrated in all experimental settings. Finally, we demonstrated that interaction of macrophage-derived S100A4 with Mel results in increased metastatic lung colonization in vivo.     

Tumor-stroma interaction: positive feedback regulation of extracellular matrix metalloproteinase inducer (EMMPRIN) expression and matrix metalloproteinase-dependent generation of soluble EMMPRIN

Matrix metalloproteinases (MMPs) are metal-dependent endopeptidases that play pivotal roles in tumor disease progression. In many solid tumors, MMPs are indeed produced by tumor stromal cells, rather than by tumor cells. This expression pattern is, at least in part, regulated by tumor-stroma interaction via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). In vitro, recombinant EMMPRIN dose-dependently stimulated MMP-1 production by primary human fibroblast cells. Interestingly, in addition to stimulating MMP expression, EMMPRIN also induced its own gene expression. To further explore this potential positive feedback regulatory mechanism, we generated human breast cancer cells expressing different levels of EMMPRIN. Coculture of EMMPRIN-positive tumor cells with fibroblast cells resulted in a concomitant stimulation of MMP-2, MMP-9, and EMMPRIN production. This induction was EMMPRIN dependent, was further enhanced by overexpression, and was reduced by antisense suppression of EMMPRIN expression in tumor cells. Increased expression of membrane-associated EMMPRIN was accompanied by an MMP-dependent generation of a soluble form of EMMPRIN representing a proteolytic cleavage product lacking the carboxyl terminus. On the basis of these findings, we propose a model in which tumor cell-associated EMMPRIN stimulates MMPs, as well as EMMPRIN expression in tumor stroma. Increased MMP activity in tumor local environment results in proteolytic cleavage of membrane-associated EMMPRIN, releasing soluble EMMPRIN. Soluble EMMPRIN in turn acts in a paracrine fashion on stroma cells that are both adjacent and distant to tumor sites to further stimulate the production of MMPs and additional EMMPRIN, which consequently contributes to tumor angiogenesis, tumor growth, and metastasis.     

S100A4基因表达在肿瘤转移中的作用

肿瘤转移是细胞恶性的重要标志之一,有许多基因和因子都参与这一过程。对S100A4基因的研究发现,它可参与细胞周期调控、细胞增殖与分化、血管生成、细胞外基质重建等多种生命过程,调控细胞的生长和运动。在某些特定的肿瘤细胞内,它的表达含量的增加可促进肿瘤细胞发生转移,并与癌症的发生具有某些相关性,可能对人类癌症的发生具有预后作用。现就S100A4基因表达与肿瘤转移的关系进行初步的探讨,以期对癌症的临床诊断提供一些参考。     

Coexpression and nuclear colocalization of metastasis-promoting protein S100A4 and p53 without mutual regulation in colorectal carcinoma

Nuclear localization of the metastasis-associated protein S100A4 has been shown to correlate with advanced disease stage in primary colorectal carcinomas (CRC), but nuclear function and its relevance for the metastatic capacity of tumor cells is still unclear. Among several nuclear interacting protein partners suggested for S100A4, the tumor suppressor protein p53 has attracted particular interest, and previous studies suggest direct and indirect modes of interaction between the two proteins. The present study was undertaken to assess coexpression and potential interaction in CRC. TP53 mutational status and S100A4 expression were investigated in a selected series of primary CRC specimens (n = 40) and cell lines (n = 17) using DNA sequencing, western blot, and double immunostaining. Additionally, S100A4 and p53 were experimentally up- and down-regulated in vitro to assess reciprocal effects. For the first time, S100A4 and p53 coexpression was demonstrated in individual CRC cells, with nuclear colocalization as a particularly interesting feature. In contrast to previous studies, no correlation was observed between TP53 mutational status and S100A4 expression, and no evidence was obtained to support reciprocal regulation between the two molecules in the HCT116 isogenic cell line model. In conclusion, S100A4 and p53 were shown to be colocalized in individual nuclei of CRC cells, and it might be speculated whether the proteins interact in this subcellular compartment.     

Transglutaminases: key regulators of cancer metastasis

The ability to metastasize represents the most important characteristic of malignant tumors. The biological details of the metastatic process remain somewhat unknown, due to difficulties in studying tumor cell behaviour with high spatial and temporal resolution in vivo. Several lines of evidence involve transglutaminases (TGs) in the key stages of tumor progression cascade, even though the molecular mechanisms remain controversial. TG expression and activity display a different role in the primary tumor or in metastatic cells. In fact, TG expression is low in the primary tumor mass, but augmented when cells acquire the metastatic phenotype. Nevertheless, in other cases, the use of inducers of TG transamidating activity seems to contrast tumor cell plasticity, migration and invasion. In the following review, the function of TGs in cancer cell migration into the extracellular matrix, adhesion to the capillary endothelium and its basement membrane, invasion and angiogenesis is discussed.     

Detailed analysis of MIA protein by mutagenesis

MIA (melanoma inhibitory activity) has been identified as a small protein secreted by malignant melanoma cells that interacts with extracellular matrix proteins including fibronectin. These findings suggest that MIA may play a role in tumor progression and the spread of malignant melanomas by mediating detachment of cells from extracellular matrix molecules. Here, we present a detailed study on functionally important MIA domains. Using site-directed mutagenesis, amino acids important for MIA structure and/or function were determined. Amino acids conserved in SH3 domains were shown to be important for structural integrity. In addition, amino acid residues necessary for MIA function were identified. Interestingly, not all of them are conserved with respect to other members of the MIA protein family. In summary, our results lead to a better understanding of MIA function. Regulating MIA functions in vivo may provide a novel therapeutic strategy for metastatic melanoma disease.     

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

S100A4 (metastasin 1) belongs to the S100 family of Ca²⁺ 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-γ ELISPOT and cytotoxicity assays. In addition, IFN-γ 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.     

Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting

Maintenance of cell and tissue homeostasis is dependent upon the dynamic balance of cell proliferation, differentiation, and apoptosis through interactions between cells and their microenvironment. The unique prostatic cellular phenotypes are induced and maintained by interaction between epithelium and adjacent stroma through intimate intercellular signaling pathways. In this article, we summarize current advances in the tumor-stroma interaction and its biologic and therapeutic implications. We specifically emphasize current studies of the possible factors driving the "vicious cycle" between stroma and emerging prostate tumor epithelial cells that may be responsible for carcinogenesis and metastasis to bone. Stroma responds both genotypically and phenotypically to tumor epithelium upon co-culture under 3-D conditions. Likewise, the emerging carcinoma responds to stromal signals that drive progression to malignancy. A vicious cycle mediated by soluble and insoluble molecules secreted by tumor cells and stroma appear be the critical factors supporting and sustaining tumor colonization in bone. Co-targeting tumor and stroma with therapeutic agents has yielded promising results both in pre-clinical models of prostate cancer and bony metastasis and in clinical trials of patients treated with a dual tumor and stroma targeting strategies. In conclusion, understanding and targeting the interaction of the tumor and its stromal microenvironmant may improve the prognosis, reduce the suffering and increase the survival of patients with advanced cancer metastasis.     

Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells

Tumor-stroma interactions play a significant role in tumor development and progression. Alterations in the stromal microenvironment, including enhanced vasculature (angiogenesis), modified extracellular matrix composition, inflammatory cells, and dys-balanced protease activity, are essential regulatory factors of tumor growth and invasion. Differential modulation of stromal characteristics is induced by epithelial skin tumor cells depending on their transformation stage when grown as surface transplants in vivo. Tumor cells can regulate the development of a "tumor-stroma" via the aberrant expression of growth factors or induction of growth factor receptors in the stromal compartment. In this context, secretion of the hematopoietic growth factors G-CSF and GM-CSF, constituitively expressed in enhanced malignant tumors, may be good candidates for induction of a tumor stroma through their effect on inflammatory cells. Upon its induction, the tumor stroma will reciprocally influence the differentiation status of tumor cells resulting in a normalization of benign tumor epithelia and the maintenance of a malignant phenotype, respectively. In the HaCaT model for squamous cell carcinoma of the skin, stromal activation and angiogenesis are transient in pre-malignant transplants, however they remain persistent in malignant transplants where progressive angiogenesis is closely correlated with tumor invasion. While continued expression of VEGF and PDGF are associated with benign tumor phenotypes, activation of VEGFR-2 is a hallmark of malignant tumors and accompanies ongoing angiogenesis and tumor invasion. As a consequence the inhibition of ongoing angiogenesis by blocking VEGFR-2 signalling resulted in dramatically impaired malignant tumor expansion and invasion. Comparably, tumor vascularization and invasion was blocked by disturbing the balance of matrix protease activity caused by a lack of PAI-1 in the stromal cells of the knockout mouse hosts. A similar inhibition of tumor vascularization was caused by TSP-1 over-expression in skin carcinoma cells, which also blocked tumor invasion and expansion. On the other hand, when granulation tissue and angiogenesis were only transiently activated as a result of stable transfection of PDGF into non-tumorigenic HaCaT cells, the target cells formed benign, but not malignant, tumors. Collectively, these data show that tumor vascularization, providing intimate association of blood vessels with tumor cells, is a prerequisite for tumor invasion. A potential mechanism for this interrelationship may be the differential regulation of MMP-expression in tumors of different grades of malignancy. In vitro MMP expression did not discriminate between benign and malignant tumor cells unless they were co-cultured with stromal fibroblasts. However, in vivo regulation of MMP expression was clearly dependent on tumor phenotype. While MMP-1 and MMP-13 were down-regulated in benign transplants, they were persistently up-regulated in malignant ones. A tight balance between proteases and their inhibitors is crucial for both the formation and infiltration of blood vessels and for tumor cell invasion, thus again emphasizing the importance of the stromal compartment for the development and progression of carcinomas.     

Cancer cell invasion is enhanced by applied mechanical stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.     

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.     

Presentation of galectin-1 by extracellular matrix triggers T cell death

Apoptotic elimination of T cells at sites of inflammation or infiltration into tumors limits an effective immune response. T cell apoptosis can be initiated by a variety of triggers, including galectin-1, a soluble, secreted lectin that binds to oligosaccharide ligands on cell surface glycoproteins, or to oligosaccharide ligands on extracellular matrix glycoproteins in tissue stroma. Although galectin-1 has no transmembrane domain and is secreted from cells that make it, it is not clear if galectin-1 functions as a soluble death trigger in vivo. We examined the ability of stromal cells secreting galectin-1 to kill T cells. Although the stromal cells synthesized abundant galectin-1, the majority of the galectin-1 remained bound to the cell surface, and stromal cell-associated galectin-1 killed bound T cells. In contrast, insufficient amounts of functional galectin-1 were released from the stromal cells into the media to kill T cells in the absence of contact with stromal cells. However, when stromal cells were grown on Matrigel, a mixture of extracellular matrix proteins, or on permeable membranes above Matrigel, secreted galectin-1 bound to Matrigel and killed T cells without stromal cell contact. Ten-fold less galectin-1 on Matrigel was sufficient to kill adherent T cells compared with soluble galectin-1. These results demonstrate that galectin-1 in extracellular matrix is able to directly kill susceptible T cells. Because increased galectin-1 deposition in tumor stroma occurs with tumor progression in various types of cancer, galectin-1 in stroma may act locally in the apoptotic elimination of infiltrating T cells during an immune response.