Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14-22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Inhibition of Homophilic Interactions and Ligand Binding of the Receptor for Advanced Glycation End Products by Heparin and Heparin-Related Carbohydrate Structures.

Background: Heparin and heparin-related sulphated carbohydrates inhibit ligand binding of the receptor for advanced glycation end products (RAGE). Here, we have studied the ability of heparin to inhibit homophilic interactions of RAGE in living cells and studied how heparin related structures interfere with RAGE⁻ligand interactions. Methods: Homophilic interactions of RAGE were studied with bead aggregation and living cell protein-fragment complementation assays. Ligand binding was analyzed with microwell binding and chromatographic assays. Cell surface advanced glycation end product binding to RAGE was studied using PC3 cell adhesion assay. Results: Homophilic binding of RAGE was mediated by V1- and modulated by C2-domain in bead aggregation assay. Dimerisation of RAGE on the living cell surface was inhibited by heparin. Sulphated K5 carbohydrate fragments inhibited RAGE binding to amyloid ß-peptide and HMGB1. The inhibition was dependent on the level of sulfation and the length of the carbohydrate backbone. α-d-Glucopyranosiduronic acid (glycyrrhizin) inhibited RAGE binding to advanced glycation end products in PC3 cell adhesion and protein binding assays. Further, glycyrrhizin inhibited HMGB1 and HMGB1 A-box binding to heparin. Conclusions: Our results show that K5 polysaccharides and glycyrrhizin are promising candidates for RAGE targeting drug development.

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons.

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans.

The current dogma in neural regeneration research implies that chondroitin sulfate proteoglycans (CSPGs) inhibit plasticity and regeneration in the adult central nervous system (CNS). We argue that the role of the CSPGs can be reversed from inhibition to activation by developmentally expressed CSPG-binding factors. Heparin-binding growth-associated molecule (HB-GAM; also designated as pleiotrophin) has been studied as a candidate molecule that might modulate the role of CSPG matrices in plasticity and regeneration. Studies in vitro show that in the presence of soluble HB-GAM chondroitin sulfate (CS) chains of CSPGs display an enhancing effect on neurite outgrowth. Based on the in vitro studies, we suggest a model according to which the HB-GAM/CS complex binds to the neuron surface receptor glypican-2, which induces neurite growth. Furthermore, HB-GAM masks the CS binding sites of the neurite outgrowth inhibiting receptor protein tyrosine phosphatase sigma (PTPσ), which may contribute to the HB-GAM-induced regenerative effect. In vivo studies using two-photon imaging after local HB-GAM injection into prick-injury of the cerebral cortex reveal regeneration of dendrites that has not been previously demonstrated after injuries of the mammalian nervous system. In the spinal cord, two-photon imaging displays HB-GAM-induced axonal regeneration. Studies on the HB-GAM/CS mechanism in vitro and in vivo are expected to pave the way for drug development for injuries of brain and spinal cord.

AMIGO2 modulates T cell functions and its deficiency in mice ameliorates experimental autoimmune encephalomyelitis.

The immune function of AMIGO2 is currently unknown. Here, we revealed novel roles of AMIGO2 in modulating T-cell functions and EAE using Amigo2-knockout (AMG2KO) mice. Amigo2 was abundantly expressed by murine T helper (Th) cells. Its deficiency impaired transplanted T-cell infiltration into the secondary lymphoid organs and dampened Th-cell activation, but promoted splenic Th-cell proliferation and abundancy therein. AMG2KO Th cells had respectively elevated T-bet in Th1- and GATA-3 in Th2-lineage during early Th-cell differentiation, accompanied with increased IFN-γ and IL-10 but decreased IL-17A production. AMG2KO mice exhibited ameliorated EAE, dampened spinal T-cell accumulation, decreased serum IL-17A levels and enhanced splenic IL-10 production. Adoptive transfer of encephalitogenic AMG2KO T cells induced milder EAE and dampened spinal Th-cell accumulation and Tnf expression. Mechanistically, Amigo2-overexpression in 293T cells dampened NF-kB transcriptional activity, while Amigo2-deficiency enhanced Akt but suppressed GSK-3ß phosphorylation and promoted nuclear translocations of NF-kB and NFAT1 in Th-cells. Collectively, our data demonstrate that AMIGO2 is important in regulating T-cell functions and EAE, and may be harnessed as a potential therapeutic target for multiple sclerosis.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix.

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation.

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

AMIGO-Kv2.1 Potassium Channel Complex Is Associated With Schizophrenia-Related Phenotypes.

The enormous variability in electrical properties of neurons is largely affected by a multitude of potassium channel subunits. Kv2.1 is a widely expressed voltage-dependent potassium channel and an important regulator of neuronal excitability. The Kv2.1 auxiliary subunit AMIGO constitutes an integral part of the Kv2.1 channel complex in brain and regulates the activity of the channel. AMIGO and Kv2.1 localize to the distinct somatodendritic clusters at the neuronal plasma membrane. Here we have created and characterized a mouse line lacking the AMIGO gene. Absence of AMIGO clearly reduced the amount of the Kv2.1 channel protein in mouse brain and altered the electrophysiological properties of neurons. These changes were accompanied by behavioral and pharmacological abnormalities reminiscent of those identified in schizophrenia. Concomitantly, we have detected an association of a rare, population-specific polymorphism of KV2.1 (KCNB1) with human schizophrenia in a genetic isolate enriched with schizophrenia. Our study demonstrates the involvement of AMIGO-Kv2.1 channel complex in schizophrenia-related behavioral domains in mice and identifies KV2.1 (KCNB1) as a strong susceptibility gene for schizophrenia spectrum disorders in humans.

Amigo adhesion protein regulates development of neural circuits in zebrafish brain.

The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion.

RAGE-mediated cell signaling.

RAGE (receptor for advanced glycation end products) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of transmembrane proteins. RAGE binds AGEs (advanced glycation end products), HMGB1 (high-mobility group box-1; also designated as amphoterin), members of the S100 protein family, glycosaminoglycans and amyloid ß peptides. Recent studies using tools of structural biology have started to unravel common molecular patterns in the diverse set of ligands recognized by RAGE. The distal Ig domain (V1 domain) of RAGE has a positively charged patch, the geometry of which fits to anionic surfaces displayed at least in a proportion of RAGE ligands. Association of RAGE to itself, to HSPGs (heparan sulfate proteoglycans), and to Toll-like receptors in the cell membrane plays a key role in cell signaling initiated by RAGE ligation. Ligation of RAGE activates cell signaling pathways that regulate migration of several cell types. Furthermore, RAGE ligation has profound effects on the transcriptional profile of cells. RAGE signaling has been mainly studied as a pathogenetic factor of several diseases, where acute or chronic inflammation plays a role. Recent studies have suggested a physiological role for RAGE in normal lung function and in neuronal signaling.

Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function.

Kainate-type glutamate receptors (KARs) regulate synaptic transmission and neuronal excitability via multiple mechanisms, depending on their subunit composition. Presynaptic KARs tonically depress glutamatergic transmission during restricted period of synapse development; however, the molecular basis behind this effect is unknown. Here, we show that the developmental and cell-type specific expression pattern of a KAR subunit splice variant, GluK1c, corresponds to the immature-type KAR activity in the hippocampus. GluK1c localizes to dendritic contact sites at distal axons, the distal targeting being promoted by heteromerization with the subunit GluK4. Presynaptic expression of GluK1c strongly suppresses glutamatergic transmission in cell-pairs in vitro and mimics the immature-type KAR activity at CA3-CA1 synapses in vivo, at a developmental stage when the endogenous expression is already downregulated. These data support a central role for GluK1c in mediating tonic inhibition of glutamate release and the consequent effects on excitability and activity-dependent fine-tuning of the developing hippocampal circuitry.

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel.

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.

Crystal structure and role of glycans and dimerization in folding of neuronal leucine-rich repeat protein AMIGO-1.

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 (high-mobility group box 1 protein) to RAGE (receptor for advanced glycation end products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons. We have determined the crystal structure of AMIGO-1 at 2.0 Å resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR-LRR interface is likely to be involved in cell-cell adhesion by AMIGO-1, while extensive glycosylation may have a role.

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons.

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.

High mobility group box-1 (HMGB1; amphoterin) is required for zebrafish brain development.

Hmgb1 (high mobility group box-1; amphoterin) is highly expressed in brain during early development of vertebrate and nonvertebrate species. However, its role in brain development remains elusive. Here we have cloned the zebrafish Hmgb1 and specifically manipulated Hmgb1 expression using injection of morpholino antisense oligonucleotides or Hmgb1 cRNA. The HMGB1 knockdown morphants produced by injection of three different morpholino oligonucleotides display a characteristic phenotype with smaller size, smaller brain width, and shorter distance between the eyes. Closer examination of the phenotype reveals severe defects in the development of the forebrain that largely lacks catecholaminergic neural networks. The HMGB1 morphant is deficient in survival and proliferation of neural progenitors and displays fewer cell groups expressing the transcription factor Pax6a in the forebrain and aberrant Wnt8 signaling. The mechanism of HMGB1-dependent progenitor survival involves the neuronal transmembrane protein AMIGO (amphoterin-induced gene and orf), the expression of which is regulated by HMGB1 in vivo. Our data demonstrate that HMGB1 is a critical factor for brain development, enabling survival and proliferation of neural progenitors that will form the forebrain structures.

Regulation of monocyte migration by amphoterin (HMGB1).

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.

Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A.

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.

AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats.

Ordered differential display identified a novel sequence induced in neurons by the neurite-promoting protein amphoterin. We named this gene amphoterin-induced gene and ORF (AMIGO), and also cloned two other novel genes homologous to AMIGO (AMIGO2 and AMIGO3). Together, these three AMIGOs form a novel family of genes coding for type I transmembrane proteins which contain a signal sequence for secretion and a transmembrane domain. The deduced extracellular parts of the AMIGOs contain six leucine-rich repeats (LRRs) flanked by cysteine-rich LRR NH2- and COOH-terminal domains and by one immunoglobulin domain close to the transmembrane region. A substrate-bound form of the recombinant AMIGO ectodomain promoted prominent neurite extension in hippocampal neurons, and in solution, the same AMIGO ectodomain inhibited fasciculation of neurites. A homophilic and heterophilic binding mechanism is shown between the members of the AMIGO family. Our results suggest that the members of the AMIGO protein family are novel cell adhesion molecules among which AMIGO is specifically expressed on fiber tracts of neuronal tissues and participates in their formation.

Enzymatic properties and intracellular localization of the novel Trichoderma reesei beta-glucosidase BGLII (cel1A).

This paper describes the characterization of an intracellular beta-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific beta-glucosidase, having no beta-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this beta-glucosidase.

Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis.

Amphoterin has been suggested to regulate invasive process extension and cell migration in tumor cells and embryonic neurons through binding to receptor for advanced glycation end products (RAGE), a multiligand transmembrane receptor belonging to the immunoglobulin superfamily. In this study, we identify a COOH-terminal motif in amphoterin (amino acids 150-183) that is responsible for RAGE binding. We show that as a surface-bound ligand, this part of amphoterin is sufficient to induce RAGE-dependent process extension, suggesting a role in the regulation of cell motility. When applied in solution, the RAGE-binding COOH-terminal motif of amphoterin efficiently inhibits process extension and transendothelial migration of tumor cells. Furthermore, in an in vivo model, the corresponding synthetic peptide significantly suppresses formation of lung metastases. Taken together, these results suggest that amphoterin binds to RAGE through a COOH-terminal motif that can be used as an efficient inhibitor to block invasive migration of tumor cells.