Invasive Properties Of Tumor Cells Research Articles

NEDD9/Arf6-dependent endocytic trafficking of matrix metalloproteinase 14: a novel mechanism for blocking mesenchymal cell invasion and metastasis of breast cancer.

NEDD9 is an established marker of invasive and metastatic cancers. NEDD9 downregulation has been shown to dramatically reduce cell invasion and metastasis in multiple tumors. The mechanisms by which NEDD9 regulates invasion are largely unknown. In the current study, we have found that NEDD9 is required for MMP14 enzymatic recovery/recycling through the late endosomes to enable disengagement of tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and tumor invasion. Depletion of NEDD9 decreases targeting of the MMP14/TIMP2 complex to late endosomes and increases trafficking of MMP14 from early/sorting endosomes back to the surface in a small GTPase Arf6-dependent manner. NEDD9 directly binds to Arf6-GAP, ARAP3, and Arf6 effector GGA3 thereby facilitating the Arf6 inactivation required for MMP14/TIMP2 targeting to late endosomes. Re-expression of NEDD9 or a decrease in Arf6 activity is sufficient to restore MMP14 activity and the invasive properties of tumor cells. Importantly, NEDD9 inhibition by Vivo-Morpholinos, an antisense therapy, decreases primary tumor growth and metastasis in xenograft models of breast cancer. Collectively, our findings uncover a novel mechanism to control tumor cells dissemination through NEDD9/Arf6-dependent regulation of MMP14/TIMP2 trafficking, and validates NEDD9 as a clinically relevant therapeutic target to treat metastatic cancer.

Regulation of membrane-type 1 matrix metalloproteinase expression by zonula occludens-2 in human lung cancer cells

During tumor invasion, tumor epithelial cells acquire migratory and invasive properties involving important phenotypic alterations. Among these changes, one can observe reorganization or a loss of cell-cell adhesion complexes such as tight junctions (TJs). TJs are composed of transmembrane proteins (occludin, claudins) linked to the actin cytoskeleton through cytoplasmic adaptor molecules including those of the zonula occludens family (ZO-1, -2, -3). We here evaluated the potential role of ZO-2 in the acquisition of invasive properties by tumor cells. In vivo, we showed a decrease of ZO-2 expression in bronchopulmonary cancers, with a preferential localization in the cytoplasm. In addition, in vitro, the localization of ZO-2 varied according to invasive properties of tumor cells, with a cytoplasmic localization correlating with invasion. In addition, we demonstrated that ZO-2 inhibition increases invasive and migrative capacities of invasive tumor cells. This was associated with an increase of MT1-MMP. These results suggest that ZO-2, besides its structural role in tight junction assembly, can act also as a repressor of tumor progression through its ability to reduce the expression of tumor-promoting genes in invasive tumor cells.

The Use of Cytokines and Chemokines in the Cancer Immunotherapy

The response of the body to cancer is not a unique mechanism and has many parallels with inflammation and wound healing. Unresolved inflammation generates a microenvironment favorable for cellular transformation and the growth of cancer cells. Chronic tissue damage triggers a repair response that includes the production of growth factors, cytokines and chemokines. Cytokines and chemokines have a crucial role in cancer-related inflammation with consequent, direct and indirect effects on the proliferative and invasive properties of tumor cells. In view of the multifactorial functions of cytokines and chemokines in tumorigenesis, the elucidation of their roles will further advance our understanding of the patho-physiological processes of tumor development and highlights potential innovative anti-cancer strategies. Despite recent advances, main anti-cancer therapies, namely surgery, radiation therapy and chemotherapy, are limited in their ability to treat minimal and metastatic residual disease. Furthermore, the benefit of conventional therapies is often limited by collateral damage to normal tissues. Immunotherapy is a new avenue of cancer treatment being investigated by researchers and clinicians for different cancer types. The aim of this paper is to analyze the recent patents and scientific reviews on the major cytokine/chemokine pathways involved in cancer immunotherapy and discuss their basic biology, clinical relevance and potential directions for future anti-cancer therapeutic applications.

Abstract A66: NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling

Abstract The adhesion scaffolding protein NEDD9 was identified as potential pro-metastatic gene in several cancers. The molecular mechanisms of NEDD9-driven metastasis are still unknown. In this study, we show that expression of NEDD9 positively correlates with the invasive stage of breast cancer. We show that NEDD9 localizes to invadopodia and endosomes. Notably, NEDD9 depleted cells have increased levels of inactive surface receptors due to increase in fast recycling of Rab4 and Rab5 positive vesicles. Mechanistically, we found that NEDD9 binds to and scaffolds the Arf6 specific GAP - ASAP3, decreasing Arf6 activity. Thus, depletion of NEDD9 leads to activation of Arf6. Inhibition of Arf6 or re-expression of NEDD9 in shNEDD9 cells was sufficient to restore recycling rates, decrease the number of Rab4 and 5 positive vesicles and the invasive properties of tumor cells. Thus, in this work, we uncover the mechanistic basis of NEDD9-driven invasion and identify a new role for NEDD9 in Arf6-dependant endocytosis. Citation Format: Elena N. Pugacheva, Yuriy Loskutov, Sarah McLaughlin, Polina Kozyulina, Varvara Kozyreva, Ryan Ice, Mark Culp, Robert Wysolmerski, Scott Weed, Alexey Ivanov. NEDD9 promotes cell invasion through modulation of ARF6 activity and endocytic recycling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A66.

Glioma-related edema: new insight into molecular mechanisms and their clinical implications

Glioma-related edema (GRE) is a significant contributor to morbidity and mortality from glioma. GRE is a complicated process involving not only peritumoral edema but also the water content of the tumor body. In terms of etiology, this condition derives from both GRE in the untreated state and GRE secondary to clinical intervention, and different cell types contribute to distinct components of GRE. Peritumoral edema was previously believed to loosen glioma tissue, facilitating tumor-cell invasion; however, the nutrition hypothesis of the tumor microecosystem suggests that tumor cells invade for the sake of nutrition. Edema is the pathologic consequence of the reconstructed trophic linkage within the tumor microecosystem. Glioma cells induce peritumoral brain edema via an active process that supplies a suitable niche for peritumoral invasive cells, suggesting that glioma-related peritumoral brain edema is determined by the invasive property of tumor cells. There are differences between pivotal molecular events and reactive molecular events in the development of GRE. Molecular therapy should target the former, as targeting reactive molecular events will produce undesired or even adverse results. At present, brain glioma angiogenesis models have not been translated into a new understanding of the features of brain images. The effect of these models on peritumoral brain edema is unclear. Clinical approaches should be transformed on the basis of new knowledge of the molecular mechanism underlying GRE. Exploring clinical assessment methods, optimizing the existing control strategy of GRE, and simultaneously developing new treatments are essential.

Chemoprevention of Colon Cancer by iNOS-Selective Inhibitors

Nitric oxide (NO) is a short-lived pleiotropic regulator and is required for numerous pathophysiological functions, including macrophage-mediated immunity and cancer. It is a highly reactive free radical produced from l-arginine by different isoforms of NO synthases (NOSs). Sustained induction of inducible NOS (iNOS) during chronic inflammatory conditions leads to the formation of reactive intermediates of NO, which are mutagenic and cause DNA damage or impairment of DNA repair, alter cell signaling, and promote proinflammatory and angiogenic properties of the cell, thus contributing to carcinogenesis. Besides its well-established role in inflammation, increased expression of iNOS has been observed in colorectal tumors and other cancers. NO-related signaling pathways involved in colon tumorigenesis seem to progress through stimulation of proinflammatory cytokines and via posttranslational protein modifications of important antiapoptotic molecules in the tumors. NO can stimulate and enhance tumor cell proliferation by promoting invasive, angiogenic, and migratory activities. In contrast, studies also suggest that high levels of NO may be protective against tumor growth by inducing tumor cell death. However, a number of in vitro studies and particularly experimental animal data support the notion that NO and its reactive metabolite peroxynitrite stimulate cyclooxygenase-2 activity, leading to generation of prostaglandins that enhance tumor growth. These prostaglandins further augment tumor promotion and invasive properties of tumor cells. Hence, selective inhibitors of iNOS and combination strategies to inhibit both iNOS and cyclooxygenase-2 may have a preventive role in colon cancer.

β1 integrin is required for anchorage-independent growth and invasion of tumor cells in a context dependent manner

Recent studies suggest that extracellular matrix (ECM) components within the tumor microenvironment can influence malignant progression, thus we investigated the influence of the ECM binding receptor β1 integrin, on the hallmark properties of tumorigenesis. Small interfering (si) or short hairpin (sh) RNA approaches were used to deplete β1 integrin in cancer cell lines. β1 integrin-depleted cells were then assessed for their growth and invasive capabilities using 2-dimensional (2D) or 3D culture conditions. Depletion of β1 integrin expression did not impact cell growth in 2D assay systems; however, β1 integrin and its ligand fibronectin were required for growth in 3D. β1 integrin-depleted cells also had reduced invasive capabilities, in part due to increased tissue inhibitor of metalloprotease (TIMP)-2 expression in conjunction with down-regulation of matrix metalloprotease (MMP)-9 levels in β1 integrin-depleted cells. Our results suggest that despite no apparent effect on 2D cell growth, fibronectin-β1 integrin signaling is a critical mediator of the 3D growth and invasive properties of tumor cells. These observations highlight the importance of investigating the role of adhesion molecules in the appropriate context and furthermore identify β1 integrin as a possible therapeutic target to inhibit the aggressive growth and invasion of tumor cells.

Secretory Leukocyte Protease Inhibitor (SLPI) Expression and Tumor Invasion in Oral Squamous Cell Carcinoma

Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies.

Abstract LB-36: Circulating epithelial tumor cells are responsive to BMP-2

Abstract Aims: Bone morphogenetic proteins (BMP) play an important role in early embryonic development as well as in organ homeostasis and in tumorigenesis. We could show previously that BMP-2 enhance migratory and invasive properties of tumor cells. Furthermore, BMP-2 affects the expression of genes which are involved in the regulation of apoptosis. A crucial step in metastasis formation is the liberation of tumor cells from the primary site, the entrance into circulation and the long-lasting persistence in the peripheral blood. Therefore we asked whether BMPs might contribute to the vitality of circulating tumor cells. Methods: Leukocytes and circulating epithelial cells from the peripheral blood of breast cancer patients were labelled with fluorescent antibodies and magnetically enriched with the MACS® system. After separation of single vital cells with the aureka® device, a single cell multiplex RT PCR was performed using the AmpliGrid system. The PCR products were analysed by urea-polyacrylamide gel electrophoresis. For real-time single cell PCR demonstrating the expression level of selected genes the Fastlane system (Qiagen, Hilden) was used. Localization of phosphorylated receptor Smads was analysed by laser scanning microscopy after one hour incubation with 100 ng/ml BMP-2. Results: Circulating epithelial cells were identified by using the cell surface antigen EpCam. Vital EpCam-positive cells were picked and subjected to multiplex RT-PCR covering 5 components of the BMP signaling cascade as well as RPL13A for control. 121 of 265 selected cells expressed RPL13A. These cells were used for further analysis. Most of these cells expressed BMP-2 (54%), BMPR-IA (46%), BMPR-IB (82%). Transcripts of the type II receptor BMPR-II (35%) and the putative BMP antagonist BMP-3 (25%) were presen to a lower extend. The expression level of BMP-2 was comparable in individual EpCam-positive cells, thus at least an autocrine stimulation of the BMP signaling pathway can be assumed. For studying the responsiveness of circulating epithelial cells towards a BMP stimulus these cells were enriched from peripheral blood and incubated with BMP-2. Vital cells showed a predominant nuclear staining for pSmad1 demonstrating a functional BMP signalling network. Conclusion: We show that key components of the BMP signaling pathway are expressed in leukocytes and epithelial cells in the peripheral blood of breast cancer patients. This indicates that the BMP signaling network potentially contributes to the activity and survival of circulating epithelial cells. This work was supported by the Dr. Rainald-Stromeyer-Stiftung Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-36. doi:10.1158/1538-7445.AM2011-LB-36

MicroRNAs as new therapeutic targets and tools in cancer

Introduction: MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level. Several studies have provided evidence that abnormal expression of selected miRNAs is associated with the pathogenesis of cancer. As they can act as either oncogenes or tumor suppressors, miRNAs have been proposed as potential new therapeutic targets or tools for cancer therapy.Areas covered: This paper reviews a significant body of the experimental data collected to date which indicate that specific miRNA inhibition or replacement can successfully modify the proliferative and invasive properties of tumor cells. It discusses recent evidence that has also revealed a direct involvement of miRNAs in drug resistance, underlying an entirely new mechanism by which tumor cells may be refractory to the treatment with cytotoxic agents. Based on these findings, in the therapeutic setting, interference with cancer-specific miRNAs could be exploited not only to produce a direct anticancer effect but also to improve the response of tumor cells to conventional treatments.Expert opinion: Overall, manipulation of miRNA functions, either by mimicking or inhibiting them, is emerging as a highly promising therapeutic strategy. However, before miRNA-based therapeutics enters the clinical armamentarium, important issues concerning specific delivery to cells/tissues of interest, safety as well as pharmacokinetic profiles needs to be addressed.

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

The YSNSG cyclopeptide derived from tumstatin inhibits tumor angiogenesis by down‐regulating endothelial cell migration

We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a beta-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained beta-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered beta1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125(FAK)), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.

Zinc‐finger transcription factor Snail accelerates survival, migration and expression of matrix metalloproteinase‐2 in human bone mesenchymal stem cells

Although bone mesenchymal stem cells (BMSC) hold promise in gene therapy and tissue engineering, the inefficient migration and the low capability of subsequent survival of BMSC have largely restrained progress in these studies. Characteristics shared between stem cells and tumorigenic cells prompted us to investigate whether mechanisms of tumor progression contribute to stem cell migration. The transcription factor Snail which functions in epithelial-mesenchymal transitions (EMT) is responsible for the acquisition of motile and invasive properties of tumor cells. It is not yet known whether Snail acts in the mechanisms of stem cell migration. Here it is shown that ectopic Snail expression increased the migration of BMSC in vitro by a mechanism dependent on the phosphoinositide 3-kinase (PI-3K) signaling pathway. Snail expression may contribute to the constitutive activation of signaling pathways of PI-3K and MAPK and the related MMP-2 secretion in BMSC. Furthermore, the stem cells expressing Snail were protected from the apoptosis triggered by serum deprivation. These results suggested the possibility for us to optimize the migration of BMSC toward infarcted tissues and their subsequent survival in the local microenvironment, by investigating mechanisms associated with the acquisition of invasiveness by tumor cells.

Ectopic expression of L-plastin in human tumor cells: Diagnostic and therapeutic implications

The process of metastasis is complex consisting of a series of sequential steps. Initially, tumor cells detach from the primary tumor, migrate through the surrounding tissue and eventually invade the lymphatic system or blood vessels. As a next step, circulating tumor cells temporarily adhere to endothelial cells and then extravasate by infiltrating the underlying basement membrane. Finally, cells migrate to a suitable location where they proliferate and form metastases (Nicolson, 1988; Stetler-Stevenson et al., 1993; Engers and Gabbert, 2000; Orr et al., 2000). Accordingly, the capacity of tumor cells to form metastatic foci correlates with the ability to proteolytically degrade basement membrane barriers and to adhere to and migrate through extracellular matrix layers (StetlerStevenson et al., 1993; Engers and Gabbert, 2000). Modulation of the actin cytoskeleton is critical for tumor cell migration and invasion (Ridley et al., 2003). Therefore, actin-binding proteins which regulate this modulation may be valuable targets to inhibit the invasive properties of tumor cells and, hence, reduce metastasis. Changes in the actin cytoskeleton are accomplished by a variety of actin-binding proteins such as cofilin, a-actinin, filamin, fascin and the plastins (Ridley et al., 2003; Samstag et al., 2003). Interestingly, the hematopoetic isoform of the plastins, L-plastin, is not only expressed by hematopoetic cells, but also by most human cancer cell lines (Lin et al., 1993a; Park et al., 1994). Here, we describe the current knowledge about the expression and function of L-plastin in human cancer cells and its implications for the therapy of cancer. ARTICLE IN PRESS

Cell Membrane-Associated MT1-MMP Dependent Activation of MMP-2 in SiHa (Human Cervical Cancer) Cells

Among the soluble MMPs, MMP-2 (gelatinase A) is particularly important in the invasive property of tumor cells. Cell membrane-associated MMP-2 activation is one of the challenging areas in tumor biology. In the present communication, we studied the membrane dependent activation of MMP-2 in SiHa cells. Activation of pro-MMP-2 by membrane fraction, membrane extract, and live SiHa cells was studied by gelatin zymography. The role of MT1-MMP in MMP-2 activation was studied by incubating SiHa cells and cell membrane fractions with anti-MT1-MMP antibody. Activation of purified pro-MMP-2 by membrane fraction isolated from SiHa cells, by SiHa cell membrane extract and by SiHa cells, pro-MMP-2 from Con A treated HT-1080 conditioned medium by SiHa cells, and pro-MMP-2 from serum free culture medium of SiHa cells and cervical tissue homogenate by SiHa cell membrane fraction was shown by gelatin zymography. SiHa membrane fraction activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture, indicating that the activation is specific for MMP-2. Inhibition of MMP-2 activation in the presence of anti-MT1-MMP antibody strongly indicated that the cell membrane mediated MMP-2 activation is MT1-MMP dependent. Immunocytochemistry of SiHa cells demonstrated expression of MT1-MMP at focal points. Invasion assay showed that invasiveness of anti-MT1-MMP antibody treated SiHa cells through Matrigel was drastically reduced compared to control SiHa cells. Our findings furnish an example of the cell membrane-associated MT1-MMP mediated MMP-2 activation in SiHa cells and suggest that this MT1-MMP mediated MMP-2 activation is of importance in tumor invasion and metastasis. This MT1-MMP mediated MMP-2 activation on tumor cell surface could be a realistic target for managing metastatic diseases.

Glycosaminoglycans modulate C6 glioma cell adhesion to extracellular matrix components and alter cell proliferation and cell migration

BackgroundAdhesion to extracellular matrix (ECM) components has been implicated in the proliferative and invasive properties of tumor cells. We investigated the ability of C6 glioma cells to attach to ECM components in vitro and described the regulatory role of glycosaminoglycans (GAGs) on their adhesion to the substrate, proliferation and migration.ResultsECM proteins (type IV collagen, laminin and fibronectin) stimulate rat C6 glioma cell line adhesion in vitro, in a dose-dependent manner. The higher adhesion values were achieved with type IV collagen. Exogenous heparin or chondroitin sulfate impaired, in a dose-dependent manner the attachment of C6 glioma cell line to laminin and fibronectin, but not to type IV collagen. Dextran sulfate did not affect C6 adhesion to any ECM protein analyzed, indicating a specific role of GAGs in mediating glioma adhesion to laminin and fibronectin. GAGs and dextran sulfate did not induce C6 glioma detachment from any tested substrate suggesting specific effect in the initial step of cell adhesion. Furthermore, heparin and chondroitin sulfate impaired C6 cells proliferation on fibronectin, but not on type IV collagen or laminin. In contrast, both GAGs stimulate the glioma migration on laminin without effect on type IV collagen or fibronectin.ConclusionThe results suggest that GAGs and proteoglycans regulate glioma cell adhesion to ECM proteins in specific manner leading to cell proliferation or cell migration, according to the ECM composition, thus modulating tumor cell properties.

Interaction between TFF1, a Gastric Tumor Suppressor Trefoil Protein, and TFIZ1, a Brichos Domain-Containing Protein with Homology to SP-C

TFF1 is a gastric tumor suppressor that protects gastric epithelial cells from damage but can promote invasive properties of tumor cells. Antibodies were raised against correctly folded TFF1 protein. These showed that the 6.67 kDa secreted trefoil protein is present as an approximately 25 kDa complex in normal human gastric mucosa. The TFF1 complex was immunopurified from human gastric mucosa and shown to comprise two proteins joined by a disulfide bond. Both were identified by amino-terminal sequencing and MALDI TOF mass spectrometry. The TFF1 protein partner is a previously unknown protein that we have called TFIZ1 for trefoil factor interactions(z) 1. TFIZ1 is expressed and secreted in normal gastric mucosa. TFIZ1 mRNA was cloned from gastric mucosa and sequenced. TFIZ1 is an 18.31 kDa protein and contains an approximately 100 amino acid brichos domain and homology with smart00019.10, SF_P. This is the first demonstration that a member of the trefoil factor family of proteins is bound covalently to a brichos domain-containing protein. The apparent molecular mass of the TFF1:TFIZ1 heterodimer is remarkably close to the theoretical molecular mass of 24.98 kDa. In conclusion, the heterodimer comprises one molecule each of TFF1 and TFIZ1, and the disulfide bond between TFF1 and TFIZ1 is the most important factor stabilizing the heterodimer.

S100A10, annexin A2, and annexin a2 heterotetramer as candidate plasminogen receptors

The defining characteristic of a tumor cell is its ability to escape the constraints imposed by neighboring cells, invade the surrounding tissue and metastasize to distant sites. This invasive property of tumor cells is dependent on activation of proteinases at the cell surface. The serine proteinase plasmin is one of the key proteinases that participate in the pericellular proteolysis associated with the invasive program of tumor cells. The assembly of plasminogen and tissue plasminogen activator at the endothelial cell surface or on the fibrin clot provides a focal point for plasmin generation and therefore plays an important role in maintaining blood fluidity and promoting fibrinolysis. S100A10, a member of the S100 family of Ca2+-binding proteins, is a dimeric protein composed of two 11 kDa subunits. Typically, S100A10 is found in most cells bound to its annexin A2 ligand as the heterotetrameric (S100A10)2(annexin A2)2 complex, AIIt. In addition to an intracellular distribution, S100A10 is present on the extracellular surface of many cells. The carboxyl-terminal lysines of S100A10 bind tPA and plasminogen resulting in the stimulation of tPA-dependent plasmin production. Carboxypeptidases cleave the carboxyl-terminal lysines of S100A10, resulting in a loss of binding and activity. Plasmin binds to S100A10 at a distinct site and the formation of the S100A10-plasmin complex stimulates plasmin autoproteolysis thereby providing a highly localized transient pulse of plasmin activity at the cell surface. The binding of tPA and plasmin to S100A10 also protects against inhibition by physiological inhibitors, PAI-1 and alpha2-antiplasmin, respectively. S100A10 also colocalizes plasminogen with the uPA-uPAR complex thereby localizing and stimulating uPA-dependent plasmin formation to the surface of cancer cells. The loss of S100A10 from the extracellular surface of cancer cells results in a significant loss in plasmin generation. In addition, S100A10 knock-down cells demonstrate a dramatic loss in extracellular matrix degradation and invasiveness as well as reduced metastasis. Annexin A2 plays an important role in plasminogen regulation by controlling the levels of extracellular S100A10 and by acting as a plasmin reductase. The mechanism by which annexin A2 regulates the extracellular levels of S100A10 is unknown. This review highlights the important part that S100A10 plays in plasmin regulation and the role this protein plays in cancer cell invasiveness and metastasis.

TaqMan ® -Based Quantification of Invasive Cells in the Chick Embryo Metastasis assay

The chick embryonic metastasis (CEM) assay is a fast in vivo method to investigate the invasive properties of tumor cells. Until now, most quantification methods were semiquantitative and time-consuming. Here we describe a rapid quantification method using TaqMan technology to quantify the invaded tumor cells in the chorioallantoic membrane of fertilized eggs. This method is based on specific detection of human ALU sequences. Moreover, it provides high sensitivity over a wide linearity range.

Development and possible clinical use of antagonists for PDGF and TGF-β

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are examples of signaling molecules which control the growth, survival motility and differentiation of cells. PDGF stimulates the growth mainly of connective tissue cells, whereas TGF-β inhibits the growth of most cell types. PDGF and TGF-β exert their cellular effects by vinding to receptors equipped with tyrosine and serine/threonine kinase activities, respectively. Both factors have important roles e.g. during the embryonal development and in wound healing.Overactivity of PDGF or PDGF receptors contributes to the development of certain diseases characterized by excessive cell growth including fibrotic disorders, atherosclerosis and malignancies. Overactivity of TGF-β also contributes to fibrotic conditions, since TGF-β promotes accumulation of extracellular matrix molecules. In cancer, TGF-β is initially a tumor suppressor due to its ability to inhibit cell growth, however, at later stages of tumor progression TGF-β has tumor promoting activity by enhancing the invasive properties of tumor cells and by suppressing the immune system and promoting angiogenesis.The involvement of PDGF in TGF-β in serious diseases makes clinically useful antagonists highly desirable. A low molecular weight receptor kinase inhibitor of the PDGF receptor kinase is now tested clinically, and TGF-β antagonists are under development. The present review discusses the development and possible clinical use of antagonsts for PDGF and TGF-β.