Multiple pathways of cell invasion are regulated by multiple families of serine proteases

The complex process of tumor invasion requires the coordinated expression and activity of cell-substratum adhesive interactions and of cell-associated protease systems, which destroy the extracellular matrix (ECM), in order to enable the invading cells to simultaneously grip and destroy the anatomical barriers that control cell spreading. A number of data indicate that such a `grip and go' process may be performed by an enlarging series of cell membrane-associated serine proteases and serine protease receptors, which provide the invasive cells with a functional unit (the protease and its receptor), able to mediate cell-substratum adhesion through specific receptor domains, to proteolytically degrade ECM and to deliver into the cell signals that up-regulate the expression either of the protease/receptor complex, or of other adhesion molecules, such as integrins. There is evidence that some proteases and protease receptor expression are under the control of tumor hypoxia, which is the result of an imbalance in oxygen supply and demand. The urokinase-type plasminogen activator (u-PA) receptor (u-PAR) is under hypoxic control and cooperates with other serine proteases of the blood coagulation pathways that may extravasate in the tumor milieu as a result of hypoxia-simulated increase of vessel permeability. Other serine proteases and their receptors cooperate with the cell-associated fibrinolytic system to promote cell invasion. Among these, tissue factor and its ligand coagulation factor VII, thrombin and its protease-activated receptors, and type II trans-membrane serine proteases seem to play a crucial role. This Review takes into consideration the complex scenario of the single serine proteases and related receptors that are involved in cell invasion, as well as the protease receptor/adhesion molecule interplay which is necessary to focus the cell surface-driven proteolysis where adhesion provides a grip to the invading cell. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tumor cell adhesion under hydrodynamic conditions of fluid flow

Current evidence indicates that tumor cell adhesion to the microvasculature in host organs during formation of distant metastases is a complex process involving various types of cell adhesion molecules. Recent results have shown that stabilization of tumor cell adhesion to the microvascular vessel wall is a very important step for successful tumor cell migration and colonization of host organs. We are beginning to understand the influences of fluid flow and local shear forces on these adhesive interactions and cellular responses within the circulation. Mechanosensory molecules or molecular complexes can transform shear forces acting on circulating tumor cells into intracellular signals and modulate cell signaling pathways, gene expression and other cellular functions. Flowing tumor cells can interact with microvascular endothelial cells mediated mainly by selectins, but the strength of these bonds is relatively low and not sufficient for stable cell adhesions. Integrin-mediated tumor cell adhesion and changes in the binding affinity of these adhesion molecules appear to be required for stabilized tumor cell adhesion and subsequent cell migration into the host organ. Failure of the conformational affinity switch in integrins results in breaking of these bonds and recirculation or mechanical damage of the tumor cells. Various cell signaling molecules, such as focal adhesion kinase, pp60src or paxillin, and cytoskeletal components, such as actin or microtubules, appear to be required for tumor cell adhesion and its stabilization under hydrodynamic conditions of fluid flow.     

Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation

Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site.     

Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-Mediated Secretion of VEGF

Prior studies have indicated that the β4 integrin promotes mammary tumor invasion and metastasis by combining with ErbB2 and amplifying its signaling capacity. However, the effector pathways and cellular functions by which the β4 integrin exerts these effects are incompletely understood. To examine if β4 signaling plays a role during mammary tumor cell adhesion to microvascular endothelium, we have examined ErbB2-transformed mammary tumor cells expressing either a wild-type (WT) or a signaling-defective form of β4 (1355T). We report that WT cells adhere to brain microvascular endothelium in vitro to a significantly larger extent as compared to 1355T cells. Interestingly, integrin β4 signaling does not exert a direct effect on adhesion to the endothelium or the underlying basement membrane. Rather, it enhances ErbB2-dependent expression of VEGF by tumor cells. VEGF in turn disrupts the tight and adherens junctions of endothelial monolayers, enabling the exposure of underlying basement membrane and increasing the adhesion of tumor cells to the intercellular junctions of endothelium. Inhibition of ErbB2 on tumor cells or the VEGFR-2 on endothelial cells suppresses mammary tumor cell adhesion to microvascular endothelium. Our results indicate that β4 signaling regulates VEGF expression by the mammary tumor cells thereby enhancing their adhesion to microvascular endothelium.     

Cytomegalovirus-infected neuroblastoma cells exhibit augmented invasiveness mediated by beta1alpha5 integrin (VLA-5)

Previously, experimental in vivo results showed that the productively and persistently human cytomegalovirus (HCMV)-infected neuroblastoma cell line UKF-NB-4AD169 exhibits a more malignant phenotype than the non-infected variant UKF-NB-4. To prove the assumption that enhanced malignancy may be due to enhanced invasive potential of the infected cells we studied interactions of both lines with monolayers of cultured endothelial cells. UKF-NB-4AD169 cells adhered to and transmigrated through endothelial monolayer to a significantly higher extent compared with UKF-NB4. Furthermore, the adhesion of UKF-NB-4AD169 but not of UKF-NB4 resulted in focal disruption of the monolayer integrity which facilitates tumor cell transmigration. Blocking antibodies directed against the beta1 integrin chain as well as beta1alpha5 on the tumor cells specifically inhibited adhesion in a concentration-dependent manner. When UKF-NB-4 were pretreated with a beta1 integrin activating antibody, focal disruption of the endothelial integrity also occurred. These findings lead us to suggest that HCMV infection activates beta1alpha5 in the host neuroblastoma cell which in turn enables these cells to tightly adhere to endothelial cells. In the presence of the protease inhibitor phenantroline, beta1alpha5-mediated adhesion was not impaired whereas UKF-NB4AD169-mediated endothelial monolayer permeabilization was dose dependently inhibited. We conclude that human cytomegalovirus infection contributes to augmented neuroblastoma invasiveness via adhesion of activated beta1alpha5 and subsequent matrix digestion by proteases.     

Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse

Summary:  Natural killer (NK) cell cytotoxicity is mediated by multiple germ line-encoded activating receptors that recognize specific ligands expressed by tumor cells and virally infected cells. These activating receptors are opposed by NK inhibitory receptors, which recognize major histocompatibility complex class I molecules on potential targets, raising the threshold for NK cell activation. Once an abnormal cell has been detected, NK cells are the sentinel source of cytolytic mediators, such as granzymes and perforins, as well as interferon-γ, which can polarize the immune response to a T-helper 1 cell type. Activation signals are transmitted by adhesion-dependent pathways, immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathways, DAP10 ITAM-independent pathways, and by signaling through immunoreceptor tyrosine-based switch motifs. These pathways activate downstream signaling partners to trigger NK cell cytotoxicity. Some of these downstream molecules are unique to the various pathways, and some of these molecules are shared. Because of the complexity of signals involved in NK cell–target cell interaction, the generation of mice with targeted mutations in signaling molecules involved in adhesion, activation, or inhibition is essential for a precise dissection of the mechanisms regulating NK cell effector functions. Here we review recent advances in the genetic analysis of the signaling pathways that mediate NK cell killing.     

Differential effects of interleukin-1 and formylmethionylleucylphenylalanine on chemotaxis and human endothelium adhesivity for A549 tumor cells

We investigated the effects of formylmethionylleucylphenylalanine (FMLP), interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) on tumor cell chemotaxis and tumor cell/endothelial cell adhesion. Chemotaxis of A549 human lung carcinoma cells was measured as the number of tumor cells which migrated across a nitrocellulose filter in a Boyden chamber. Tumor cell/endothelial cell adhesion was measured as the number of 125IUdR tumor cells adherent to monolayers of endothelial cells. Confluent monolayers of human umbilical endothelial cells were incubated from 10 to 240 minutes with FMLP, monocyte-derived interleukin-1, or recombinant IL1 alpha or IL1 beta. The endothelial cells were washed and then incubated with 125IUdR-tumor cells. Thirty minutes later the number of adherent tumor cells was assessed isotopically. Our results demonstrate that (a) interleukin-1 but not FMLP, has chemotactic activity for tumor cells, and (b) both FMLP and interleukin-1 enhance tumor cell adhesion to the endothelium independent of any chemotactic activity. Furthermore, we demonstrate that IL1 alpha and IL1 beta have different effects on tumor cell/endothelial cell adhesion, and raise the possibility that IL1 alpha but not IL1 beta is continuously synthesized and stored within the endothelium. We postulate that IL1 alpha and IL1 beta influence tumor cell/endothelial cell adhesion independent of chemotaxis through the expression of adhesive receptors on the endothelial cell surface.     

Galectin-3 Protects Human Breast Carcinoma Cells against Nitric Oxide-Induced Apoptosis : Implication of Galectin-3 Function during Metastasis

Galectin-3 is a beta-galactoside-binding protein which regulates many biological processes including cell adhesion, migration, cell growth, tumor progression, metastasis, and apoptosis. Although the exact function of galectin-3 in cancer development is unclear, galectin-3 expression is associated with neoplastic progression and metastatic potential. Since studies have suggested that tumor cell survival in microcirculation determines the metastatic outcome, we examined the effect of galectin-3 overexpression in human breast carcinoma cell survival using the liver ischemia/reperfusion metastasis model. While the majority of control cells died by hepatic ischemia/reoxygenation, nearly all of galectin-3 overexpressing cells survived. We showed that galectin-3 inhibits nitrogen free radical-mediated apoptosis, one of the major death pathways induced during hepatic ischemia/reperfusion. Galectin-3 inhibition of apoptosis involved protection of mitochondrial integrity, inhibition of cytochrome c release and caspase activation. Taking these results together with the previous observation that galectin-3 inhibits apoptosis induced by loss of cell adhesion, we propose that galectin-3 is a critical determinant for anchorage-independent and free radical-resistant cell survival during metastasis.     

Platelet adhesion and fusion to endothelial cell facilitate the metastasis of tumor cell in hypoxia-reoxygenation condition

To investigate the relevant molecular mechanisms of platelet in promoting metastasis of tumor cell. The adhesion of fluorescence dye labeled-platelet to human liver sinusoidal endothelial cell (LSEC) line and tumor cell lines were detected by fluorescence microscope and fluorescence plate reader or laser scanning confocal microscope. The relevant adhesion molecules were analyzed by the antibody blockage experiment. The immune colloidal gold transmission electron microscope (TEM), flow cytometry and dye transfer were used to decipher the adhesion and fusion of platelet and LSEC. The tumor cells adhesion to vessels in ischemia condition was analyzed on mouse mesenteric vessels and the metastasis and neovascularization of metastatic foci in pulmonary tissue were also detected after tumor cells injected into nude mice via tail veil. After hypoxia-reoxygenation, tumor cell or LSEC markedly increased its adhesion with platelet, which could be blocked by different antibodies to platelet adhesion molecules. Platelet increased adhesion of tumor cell to LSEC in dose-dependent manner. The fusion of platelet and LSEC was demonstrated by translocation of fluorescent dye from platelet into the adherent LSEC; gpIIb emerged on the LSEC; and confirmed by TEM. The morphological examination found platelet presented between tumor cell and LSEC. Animal experiment indicated that the tumor adhesion to vessels was seldom in normal condition, but increased in ischemia-reperfusion condition, and further significantly enhanced by platelets. The number of tumor metastatic foci and the density of blood vessels within metastatic foci in lung were markedly increased by tumor cell pre-adhered with platelet. The adhesion or fusion of platelet to endothelial cell mediated by platelet surface adhesion molecules, which could promote the adhesion of tumor cell with endothelial cells and the tumor metastasis.     

Focal adhesion kinase regulates metastatic adhesion of carcinoma cells within liver sinusoids

Organ-specific tumor cell adhesion to extracellular matrix (ECM) components and cell migration into host organs often involve integrin-mediated cellular processes that can be modified by environmental conditions acting on metastasizing tumor cells, such as shear forces within the blood circulation. Since the focal adhesion kinase (FAK) appears to be essential for the regulation of the integrin-mediated adhesive and migratory properties of tumor cells, its role in early steps of the metastatic cascade was investigated using in vitro and in vivo approaches. Human colon and hepatocellular carcinoma cells were used to study adhesive properties under static conditions and in a parallel plate laminar flow chamber in vitro. In addition, intravital fluorescence microscopy was used to investigate early interactions between circulating tumor cells and the microvasculature of potential target organs in vivo. Shear forces caused by hydrodynamic fluid flow induced Tyr-hyperphosphorylation of FAK in cell monolayers. Reduced expression of FAK or its endogenous inhibition by FAK-related non-kinase (FRNK) interfered with early adhesion events to extracellular matrix components under flow conditions. In contrast, tumor cell adhesion to endothelial cells under these conditions was not affected. Furthermore, down-regulation of FAK inhibited metastatic cell adhesion in vivo within the liver sinusoids. In summary, FAK appears to be involved in early events of integrin-mediated adhesion of circulating carcinoma cells under fluid flow in vitro and in vivo. This kinase may take part in the establishment of definitive adhesive interactions that enable adherent tumor cells to resist fluid shear forces, resulting in an organ-specific formation of distant metastases.     

LIM激酶与肿瘤

LIM激酶（LIM kinase, LIMK）家族主要有两个成员：LIMK1和LIMK2,它们在癌症的侵袭和迁移过程中起着重要的作用。其通过调节cofilin家族蛋白的活性而影响肌动蛋白细胞骨架结构,对细胞形态、运动、黏附以及迁移都至关重要。LIMK对cofilin蛋白磷酸化与去磷酸化间的平衡进行微调节可能是影响肿瘤细胞迁移侵袭能力的决定性因素。LIMK受多条上游信号通路调控,主要上游信号分子是RhoGTP酶和Slingshot磷酸酶,形成Rho-ROCK/PAK-LIMK-Cofilin和SSH- LIMK-Cofilin信号通路。LIMK分布广泛并对多种组织的正常发育必不可少,在多种肿瘤组织中高表达并参与肿瘤生长、血管形成以及迁移侵袭过程。本文综述了LIMK的结构、功能、组织表达,参与肿瘤细胞迁移侵袭的机制以及信号通路的调控。     

Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung

Cancer progression is facilitated by blood coagulation. Anticoagulants, such as Hirudin and low molecular weight heparins (LMWHs), reduce metastasis mainly by inhibition of thrombin formation and L- and P-selectin-mediated cell-cell adhesion. It is unknown whether the effects are dependent on cancer cell type. The effects of anticoagulants on tumor development of K1735 and B16 melanoma cells and CT26 colon cancer cells were investigated in mouse lung. Tumor load was determined noninvasively each week up to day 21 in all experiments using bioluminescence imaging. Effects of anticoagulants on tumor development of the three cell lines were correlated with the fibrin/fibrinogen content in the tumors, expression of tissue factor (TF), protease activated receptor (PAR)-1 and -4 and CD24, a ligand of L- and P-selectins. Hirudin inhibited tumor development of B16 cells in lungs completely but did not affect tumor growth of K1735 and CT26 cells. Low molecular weight heparin did not have an effect on K1735 melanoma tumor growth either. TF and PAR-4 expression was similar in the three cell lines. PAR-1 and CD24 were hardly expressed by K1735, whereas CT26 cells expressed low levels and B16 high levels of PAR-1 and CD24. Fibrin content of the tumors was not affected by LMWH. It is concluded that effects of anticoagulants are dependent on cancer cell type and are correlated with their CD24 and PAR-1 expression.     

Modulating malignant epithelial tumor cell adhesion,migration and mechanics with nanorod surfaces

The failure of tumor stents used for palliative therapy is due in part to the adhesion of tumor cells to the stent surface. It is therefore desirable to develop approaches to weaken the adhesion of malignant tumor cells to surfaces. We have previously developed SiO₂ coated nanorods that resist the adhesion of normal endothelial cells and fibroblasts. The adhesion mechanisms in malignant tumor cells are significantly altered from normal cells; therefore, it is unclear if nanorods can similarly resist tumor cell adhesion. In this study, we show that the morphology of tumor epithelial cells cultured on nanorods is rounded compared to flat surfaces and associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell viability and proliferation was unchanged on nanorods. Adherent cell numbers were significantly decreased while single tumor cell motility was increased on nanorods compared to flat surfaces. Together, these results suggest that nanorods can be used to weaken malignant tumor cell adhesion, and therefore potentially improve tumor stent performance.     

Matrix metalloproteinase-1 and thrombin differentially activate gene expression in endothelial cells via PAR-1 and promote angiogenesis

Many tumor types express matrix metalloproteinase-1 (MMP-1); its collagenase activity facilitates both tumor cell invasion and metastasis. MMP-1 expression is also associated with increased angiogenesis; however, the exact mechanism by which this occurs is not clear. MMP-1 proteolytically activates protease activated receptor-1 (PAR-1), a thrombin receptor that is highly expressed in endothelial cells. Thrombin is also present in the tumor microenvironment, and its activation of PAR-1 is pro-angiogenic. It is currently unknown whether MMP-1 activation of PAR-1 induces angiogenesis in a similar or different manner compared with thrombin. We sought to determine the mechanism by which MMP-1 promotes angiogenesis and to compare the effects of MMP-1 with those of thrombin. Our results demonstrate that via PAR-1, MMP-1 activates mitogen-activated protein kinase signaling cascades in microvessel endothelial cells. Although thrombin activation of PAR-1 also induces signaling through these pathways, the time-course of activation appears to vary. Gene expression analysis revealed a possible consequence of these signaling differences as MMP-1 and thrombin induce expression of different subsets of pro-angiogenic genes. Furthermore, the combination of thrombin and MMP-1 is more angiogenic than either protease alone. These data demonstrate that MMP-1 acts directly on endothelial cells as a pro-angiogenic signaling molecule and also suggest that the effects of MMP-1 may complement the activity of thrombin to better facilitate angiogenesis and promote tumor progression.     

Interactions between cell adhesion and the synaptic vesicle cycle in Parkinson’s disease

Synaptic dysfunction has been identified as an early neuropathologic event in Parkinson’s disease. Synapses depend critically on the adhesion of neurons to one another, glial cells, and the extracellular matrix. Cell–cell and cell–matrix adhesions regulate the structure and function of synapses, in part, through interactions with structural elements such as actin and microtubule proteins. These proteins are critical not only for neuronal structure and polarity, but also for the synaptic vesicle cycle, including maintenance of and transfer between vesicle pools, exocytosis, and vesicle recycling. Pathway analyses of genome wide association studies (GWAS) in Parkinson’s disease have identified frequent single nucleotide polymorphisms (SNPs) in cell adhesion pathways, suggesting that dysfunction in cell adhesion may play a role in disease pathology. Based on these observations, it may be hypothesized that Parkinson’s disease is due to synaptic dysfunction caused by genetic variations in cell adhesion pathways that affect actin and/or microtubule-mediated events in the synaptic vesicle cycle. Furthermore, it is hypothesized that cells with pacemaker-like activity—a characteristic of neurons that degenerate in Parkinson’s disease—may depend more on actin for recruiting synaptic vesicles for release than do less active neurons, thereby enhancing their sensitivity to SNPs in cell adhesion pathways and explaining the selectivity of neurodegeneration. Cells may ultimately die due to detachment from the extracellular matrix. This hypothesis suggests that further exploration of cell adhesion pathways and their linkage to neurotransmitter release through cell structural proteins such as actin and microtubules may provide important insights into Parkinson’s disease.     

Molecular mechanisms of lymphocyte-mediated cytotoxicity

Granule-mediated cytotoxicity is the major mechanism for lymphocytes to kill viruses,intracellular bacteria andtumors.The cytotoxic granules move to the immunological synapse by exocytosis after recognition of a killer cell.The contents of the granules are delivered into target cells with the help of perforin by endocytosis.A group ofserine protease granzymes cleave their critical substrates to initiate DNA damage and cell death.The mostabundant granzymes are granzyme A and B.They induce cell death through alternate and nonoverlappingpathways.The substrates and functions of the majority of the orphan granzymes have not yet been identified.It ispossible that the diversity of granzymes provides fail-safe mechanisms for killing viruses and tumor cells.Cellular& Molecular Immunology.2005;2(4):259-264.     

Delivery and therapeutic potential of human granzyme B

Summary: Granzyme B (GzmB) is used by cytotoxic lymphocytes as a molecular weapon for the defense against virus-infected and malignantly transformed host cells. It belongs to a family of small serine proteases that are stored in secretory vesicles of killer cells. After secretion of these cytolytic granules during killer cell attack, GzmB is translocated into the cytosol of target cells with the help of the pore-forming protein perforin. GzmB has adopted similar protease specificity as caspase-8, and once delivered, it activates major executioner apoptosis pathways. Since GzmB is very effective in killing human tumor cell lines that are otherwise resistant against many cytotoxic drugs and since GzmB of human origin can be recombinantly expressed, its use as part of a ‘magic bullet’ in tumor therapy is a very tempting idea. In this review, we emphasize the peculiar characteristics of GzmB that make it suited for use as an effector domain in potential immunoconjugates. We discuss what is known about its uptake into target cells and the trials performed with GzmB-armed immunoconjugates, and we assess the prospects of its potential therapeutic value.