Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. Binding of p120 catenin stabilizes junctional cadherins by preventing cadherin endocytosis  is a multiprotein structure present in most organisms ranging from insects to mammals , 2; its iewed in , 10), whskeleton , 12. in vitro and in vivo [Endothelial cells play a critical role in the regulation of vasoreactivity, hemostasis, and leukocyte recruitment. Vascular endothelial cells are also critical for maintaining normal tissue function by acting as a selective barrier that regulates the passage of fluid, macromolecules, and leukocytes from the vascular space to the interstitium. The activation of proinflammatory pathways induces a loss of endothelial barrier function through activation of membrane receptors in endothelial cells, triggering several signaling cascades, including the activation of kinase signaling, small GTPase-mediated actin cytoskeleton remodeling, and calcium release (reviewed in –16). Two in vivo , 19. In α-catenin, β-catenin, and γ-catenin [β-catenin and γ-catenin phosphorylation [The development of antibodies that recognize phosphotyrosine residues quickly enabled research that demonstrated a critical role for tyrosine phosphorylation in the modulation of intercellular adhesion, in particular through the regulation of AJ-based contacts. Maher et al. were the first to show that cell-cell junctions in epithelial cells (PtK2 and MDCK) and chicken embryo fibroblasts contained proteins phosphorylated on tyrosine . Within -catenin –32. A si-catenin . Furtherrylation , demonstrylation  describerylation  and to arylation . Src wasrylation . Early rrylation . Using vrylation , 30, 38.β-catenin, and γ-catenin (but not E-cadherin) tyrosine phosphorylation was increased after calcium-induced differentiation, which coincided with an increased association of α-catenin and p120 with E-cadherin [ Drosophila, as a dominant negative mutant of the Src homolog Src42A induced the disorganization of DE-cadherin contacts [ Drosophila embryos lacking both Src homologs Src42A and Src64 showed diminished DE-cadherin and armadillo staining at cell-cell junctions [Oncogenic Src mutants may have different substrate specificity than endogenous kinases , which mcadherin . Converscadherin . Similarcadherin . Src actunctions . Togetheβ-catenin binding to N-cadherin, by promoting Y152 phosphorylation in the phosphatase PTP1B, which in turn was responsible for dephosphorylating Y654 in β-catenin [The mechanisms involved in Src-mediated AJ formation are not well understood. Tyrosine kinases may promote AJ assembly through phosphorylation of its components or indirectly via the regulation of tyrosine phosphatases as well as small GTPases. For example, Abl kinases can promote AJ formation through the regulation of Rho and Rac signaling , 45, whi-catenin . PTK sigα [α-induced Src activation in MCF7 cells [The research discussed so far has placed cadherins and catenins downstream of PTK activity, but E-cadherin engagement can also regulate PTK activity, placing cadherins correspondingly upstream of these kinases . For insα –51, presα . Cadheriα . E-cadheα  but induα  and in mα . N-cadheα . Howeverα , a pathwα , 59. Simα  and for α –63. Furtα ) from VEα . This meF7 cells , 49–51. F7 cells , 67, plaAlthough some of the molecular mechanisms are being teased out, it still remains largely unknown how cadherin association can regulate RTK signaling. An important clue comes from the VE-cadherin and VEGFR2-dependent response to shear in endothelial cells –63. Fluiβ-catenin, γ-catenin, and p120 [Similar to other classical cadherins, the phosphorylation state of VE-cadherin was found to be associated with differences in endothelial function. Lampugnani et al. first showed that a phosphotyrosine antibody labeled endothelial cell-cell contacts  by showiand p120 . VEGF aland p120 . Most imand p120 –79, demo In vitro, Src was able to directly phosphorylate a short peptide containing this residue. Other peptides containing C-terminal tyrosines (from Y645 to Y784) were not a good substrate for this in vitro assay, suggesting that either Src is not capable of directly phosphorylating these tyrosines in VE-cadherin or that other docking site(s) in the full length protein are needed for this reaction. No phosphorylation in serine was detected upon VEGF treatment [β-arrestin binding and VE-cadherin endocytosis. VE-cadherin serine 665 phosphorylation was also implicated downstream of R-Ras, a small GTPase required for vascular differentiation that is downregulated in the leaky tumor vasculature [The particular phosphorylation sites of VE-cadherin in response to VEGF are a matter of intense investigation. This question became further complicated by the lack of specificity of some commercial antibodies used in previous studies. Wallez et al.  showed treatment . This isreatment , who shoculature . In HUVEculature .α, LPS, H2O2, and high glucose [α [α- or LPS-induced increase in permeability did not require phosphatase inhibition, raising the question of whether endogenous phosphatases are sufficient to blunt the tyrosine phosphorylation induced by these agonists, without affecting the increase in permeability. In any case, a requirement of tyrosine kinase activity was demonstrated by the ability of several kinase inhibitors to block the increase in monolayer permeability induced by TNF-α or LPS [α-induced loss of barrier function than SFK-specific inhibitors PP1 and PP2, suggesting that other kinases may be involved in parallel pathways [VE-cadherin phosphorylation also occurs in response to other stimuli, including TNF- glucose –87, albeucose [α  or LPS [ucose [α  to humanα or LPS , 85. Notpathways . in vivo in the absence of any pathological condition. Lambeng et al. [ in vivo that other signals must be activated concurrently.A wealth of data unmistakably points to a very important role for SFK-mediated tyrosine phosphorylation of endothelial AJ in the regulation of barrier function. However, as in the case of epithelial cells presented above, the link is not straightforward or unidirectional. SFK activity and VE-cadherin phosphorylation can be observedg et al.  showed tg et al. . Moreoveg et al.  showed tg et al. . More reg et al.  showed tg et al. . In contg et al. , Wessel g et al.  did not g et al. . Tyrosing et al. , 93, raig et al. , 67, whig et al. . A potenIn vitro, the effect of SFK activation on human dermal microvascular cells depends on the method of activation [ In vitro assays suggested that this dephosphorylation event was due to clathrin-dependent VE-cadherin endocytosis and ubiquitin-mediated degradation, rather than a direct action of a phosphatase [tivation . Consisttivation , demonsttivation . Interessphatase .2+ release, Rho activation, actin remodeling, and tyrosine kinase activation, surrounding the leukocyte in what is called the adhesion cup and promoting the cytoskeletal changes to make room for the transmigrating leukocyte. This review will focus on tyrosine kinase signaling, and the reader is referred to recent excellent reviews [Leukocyte infiltration into inflamed tissues is a major aspect of the body's response to damage. To arrive at the required location, leukocytes must travel through the endothelium, in a process called extravasation. This is a multistep process that involves complex interactions between the leukocyte and the endothelial cell. Leukocytes bind to activated endothelium and initiate a cascade of intermolecular contacts that allow them to traverse from the bloodstream into the stroma through the endothelium via either a transcellular route  or a paracellular route (opening a gap between two adjacent ECs) . The reviews , 98, 99 α-activated HUVECs, which could be rescued by reexpression of wild-type cortactin-GFP, but not by a cortactin mutant in which three tyrosines  were mutated to phenylalanine (cortactin 3F-GFP) [α-treated HUVECs was also blocked by cortactin knockdown, PP2 treatment, or expression of either tailless ICAM-1-GFP or cortactin 3F-GFP. More importantly, cortactin siRNA blocked the clustering of actin and ICAM-1 around adherent PMN [ in vivo was provided by Schnoor et al. [ In vitro, an EPAC-specific cAMP analog rescued the increased permeability, while TEM efficiency was restored by expression of a constitutive form of RhoG [Early on, a critical role was recognized for tyrosine phosphorylation in leukocyte transendothelial migration (TEM), at least in part mediated by leukocyte integrins binding to ICAM-1, leading to remodeling of the actin cytoskeleton . ICAM-1  3F-GFP) . ICAM-1 rent PMN . Altogetr et al.  who foun of RhoG , a GTPas of RhoG .Consistent with a role for cortactin-mediated ICAM-1 clustering, apical ICAM-1 mobility was reduced in HUVECs after ICAM-1 antibody-mediated cross-linking in cells expressing full length ICAM-GFP, but not tailless ICAM-GFP . However in vitro on tyrosine 512 [α-induced ICAM-1 cleavage was abolished by Y512A mutant [ in vivo.Interestingly, leukocyte receptors can be phosphorylated on tyrosine as well . Src wassine 512 . Bindingsine 512 . A possiA mutant . In HUVEA mutant . ImportaA mutant . HoweverA mutant , suggestA mutant . ElucidaPECAM-1 is another leukocyte receptor known to be phosphorylated on at least two tyrosines, Y633 and Y686 . Contrar in vivo [ in vitro [α-treated HUVECs [α-treated HUVECs [β-catenin nor VE-cadherin was seen at sites of ICAM-1 engagement [32P-labeled CHO-ICAM-1 cells showed that ICAM-1 cross-linking promoted VE-cadherin phosphorylation at Y731. Interestingly, the authors mentioned that the majority of phosphorylation events in VE-cadherin occur at serine and threonine, rather than tyrosine residues [ in vitro was confirmed in GPNT cells, which express endogenous VE-cadherin. In these cells, expression of Y731F  significantly reduced T-cell TEM. No mutant affected lymphocyte adhesion to these cells [ in vivo [ in vivo, as suggested by the data obtained by Allingham et al. [ in vitro data show that leukocyte attachment can promote VE-cadherin phosphorylation, but in vivo experiments suggest that the critical VE-cadherin tyrosine is constitutively phosphorylated, suggesting that the main target for SFK-mediated phosphorylation downstream of leukocyte attachment  may be other proteins than VE-cadherin, such as cortactin, FAK, or eNOS.To allow TEM via the paracellular route, the endothelial cell must disassemble the adherens and tight junctions that sustain the strong homotypic intracellular contacts in order to create the space for their migration . Early w in vivo  and in vin vitro  and thatin vitro . There hin vitro  and beadd HUVECs , 117. Ind HUVECs . Similard HUVECs . Tyrosind HUVECs . Other rd HUVECs  and VCAMd HUVECs , 120. Ingagement . The rearesidues , but these cells . Critica in vivo . While tβ-catenin also increases VE-cadherin adhesion strength [α-catenin and thus the actin cytoskeleton [α-catenin and are thus independent of β-catenin association and dissociation. Expression of an E-cadherin construct fused to α-catenin promoted strong cell-cell adhesion [α-catenin chimera demonstrated that the dissociation of α-catenin from VE-cadherin is a required step for the induction of vascular permeability by VEGF or histamine and for allowing neutrophil or lymphocyte recruitment into inflamed tissues.Catenin binding is essential to support cadherin-based adhesions. Accordingly, p120 binding increases E-cadherin lateral clustering and adhesion strength , 122 andstrength  and funcskeleton , 12. A cadhesion , 132. Moadhesion  using knβ-catenin phosphorylation may function as a switch to allow or prevent cadherin association with the actin cytoskeleton by affecting its ability to bind E-cadherin and α-catenin. In F9 cells, a phosphomimetic Y142E mutation in β-catenin did not coimmunoprecipitate with α-catenin, while Y654E β-catenin mutation reduced but did not abolish the ability to coimmunoprecipitate with E-cadherin [β-catenin association with E-cadherin and α-catenin. Accordingly, in IEC18 intestinal epithelial cells, overexpression of K-Ras led to β-catenin phosphorylation at tyrosines 142 and 654, inducing the dissociation from both E-cadherin and α-catenin [β-catenin construct at tyrosine 142, leading to the dissociation of β-catenin from α-catenin but not from E-cadherin. Y142F β-catenin mutant was resistant to Fer-induced loss of α-catenin binding. In contrast, Src and Yes were able to phosphorylate β-catenin at sites other than tyrosine 142 [β-catenin at tyrosine Y142, promoting the dissociation of β-catenin from VE-cadherin [The observation that tyrosine phosphorylation regulates cell-cell adhesion, together with the finding that cadherins and catenins are targets for tyrosine kinases, led to intense research to determine how phosphorylation affected the adherens junction structure, with the notion that the phosphorylation of cadherins and/or catenins may lead to changes in cadherin/catenin association. Consistently, cadherin , suggest-catenin . Fer andsine 142 . In mouscadherin .α-catenin or β-catenin, respectively, even when v-Src induced a marked increase in E-cadherin phosphorylation. Further, v-Src activation reduced cell adhesion strength in L fibroblasts expressing an E-cadherin-α-catenin fusion construct that did not bind β-catenin, demonstrating that Src can inhibit cell adhesion independently of junction disassembly through β-catenin phosphorylation [2+ media displayed increased phosphorylation of β-catenin and γ-catenin, which correlated with an increased association of α-catenin and p120 with E-cadherin [β-catenin may be regulated independently. For example, Ras-induced transformation of MCF10A human mammary epithelial cells promoted the tyrosine phosphorylation of AJ components and a loss of E-cadherin/β-catenin binding concurrently with an increase in E-cadherin/p120 association [ in vitro phosphorylation and binding assays demonstrated that Src can directly phosphorylate p120 and β-catenin with markedly different outcomes: while β-catenin phosphorylation at Y654 reduced β-catenin affinity for an E-cadherin cytosolic domain, p120 phosphorylation increased E-cadherin binding [β-catenin binding to N-cadherin through phosphorylation and activation of the phosphatase PTP1B, which in turn was responsible for dephosphorylating β-catenin at tyrosine 654 [γ-catenin phosphorylation can also lead to different results, depending on the kinase involved and the phosphorylated tyrosine. While Src-mediated phosphorylation at tyrosine 683 reduced the association of γ-catenin with E-cadherin and α-catenin, Fer-induced phosphorylation at tyrosine 549 increased γ-catenin binding to α-catenin [Even though phosphorylation-induced loss of catenin binding is an attractive mechanism to explain the reduction of cell-cell adhesion, accumulated evidence clearly shows that AJ phosphorylation cannot be directly linked to AJ disassembly in every case. Instead, the net effect is the overall sum of multiple actions to either increase or decrease AJ protein association, depending on the kinase involved and the specific tyrosines phosphorylated. For example, in 3Y1 fibroblasts  and MDCKrylation . Keratincadherin . Furtherociation . Similarociation . Consist binding . In E8 csine 654 . γ-caten-catenin .β-catenin, respectively. Expression of the same mutants prevented the formation of a tight barrier in these cells [β-catenin and p120 binding to VE-cadherin, which correlated with phosphorylation of both VE-cadherin and β-catenin at tyrosine 654 [β-catenin and p120 [β-catenin and reduced p120 association with VE-cadherin that was associated with a loss of barrier function [β-catenin. Early on, Esser et al. [β-catenin, or γ-catenin [β-catenin, and γ-catenin was not affected [Tyrosine phosphorylation has also been linked to AJ dissociation in the endothelium. Tyrosines 658 and 731 in the VE-cadherin tail are required for binding to catenins , since pse cells . Similarse cells . Consistsine 654 , and expand p120 . Furtherfunction . VE-cadhr et al.  showed t-catenin . Moreove-catenin . Thus, m-catenin . Inhibit-catenin . While caffected . Y530A Saffected , demonstβ-catenin without affecting its association with VE-cadherin, as measured by coimmunoprecipitation [β treatment in bovine pulmonary artery cells [α [β, it appears that gap formation precedes a loss of catenin staining [ in vitro [ in vivo to ensure a fast gap closure as proposed by Baluk et al. [Thrombin, another potent agent that induces a rapid increase in endothelial permeability, promoted the tyrosine phosphorylation of pitation . In HPAEpitation . Interespitation , suggestry cells , the discells [α , and thecells [α . In thescells [α –149. In staining , suggestin vitro  and thisk et al.  after deβ-catenin and p120 phosphorylation [α was shown to mediate thrombin- and LPS-induced p120 phosphorylation at serine 879, leading to the dissociation from VE-cadherin and AJ disassembly [α-catenin, β-catenin, and γ-catenin binding, and mutation of a cluster of eight serine residues from S838 to S853 prevented E-cadherin binding to the catenins (as measured by E-cadherin IP of 35S-labeled cells) and abolished the ability of E-cadherin to promote cell aggregation, a method to determine cell-cell contact strength [β-catenin [β-catenin binding in NIH3T3 expressing exogenous mouse E-cadherin [ In vitro, CK-II phosphorylated wild-type E-cadherin, while S840A, S853A, and S855A E-cadherin mutants were resistant to CK-II-mediated phosphorylation [ in vitro [β-catenin but did not modify the association with p120 [852S853E854 → T748D749S750), arguing to support a conserved need for negative charges in this domain. CK-I is best known as a component of the β-catenin destruction complex that is part of the Wnt pathway [α-catenin. In response to Wnt3a, CK-I promoted p120 phosphorylation, linking p120 to Wnt-mediated β-catenin transcription [α-catenin at serine 641, leading to the release of β-catenin and an increase in β-catenin transactivation [α-catenin in vitro on a cluster of serine and threonine residues at the α-catenin flexible linker [α-catenin mutants in MDCK cells in a model of monolayer fragmentation suggested that phosphorylation of α-catenin at this region promotes cell-cell adhesion strength and monolayer integrity. This effect, however, was not due to changes in junctional assembly, as all mutants associated with E-cadherin or β-catenin to the same extent [The regulation of the adherens junction complex may also involve phosphorylation of serine and threonine. In HPAE cells, thrombin effects were associated with a PKC-dependent dephosphorylation of VE-cadherin and rylation . As VE-crylation . In factassembly . E-cadhestrength . Structu-catenin , 155. E-cadherin , 157. Inrylation . In anotin vitro , while Cith p120 . Serines pathway , but it cription . In resptivation . Recentle linker , includie extent .Overall, the biochemical data suggest that the observation of a particular phosphorylation event may not necessarily imply that large changes in cadherin/catenin association will be detected. While this fact does not negate an important role in junctional phosphorylation in the regulation of the adherens junction assembly and disassembly, several conclusions can be drawn from all the accumulated evidence. First, tyrosine signaling must act in concert with other pathways to promote junctional disassembly and loss of cell adhesion, as direct kinase activation does not always lead to junctional loss. Second, kinase deficient models and small molecule inhibitors demonstrated that a basal level of tyrosine phosphorylation is required for junctional maintenance and that a precise regulation of the levels of activity may be needed in order to ensure normal cell adhesion. Third, fluorescence imaging showed that the loss of cell-cell contacts is reversible and geographically limited, and thus biochemical methods may lack the sensitivity to detect small but important changes. It can be envisioned that junctional disassembly and loss of cadherin/catenin association may be strictly limited to subcellular regions immediately surrounding the formation of a gap or transmigrating leukocyte and temporally restricted to the initiation of such event, reforming quickly to allow junctional recovery.β1 association with Fyn, leading to the association of activated FAK with p120 [β-catenin after S1P treatment, and knockdown of β-catenin prevented the association between FAK and VE-cadherin. Moreover, Lyn kinase, an SFK, promoted the stabilization of endothelial barrier through phosphorylation of FAK at tyrosines 576/577 and 925 [ in vitro and ex vivo that FAK promotes junctional assembly and endothelial barrier function. Nevertheless, published data also argues in favor of another role in promoting barrier disruption. FAK knockdown in immortalized human microvascular cells increased basal TEER [ in vitro VE-cadherin at Y658 [Although most of the efforts to understand tyrosine kinase signaling in the regulation of endothelial contacts have been focused on SFKs, an important role emerged for the focal adhesion kinase family of tyrosine kinases. As its name implies, FAK is best known in the context of integrin signaling (reviewed in ). Notablith p120 . FAK wasith p120 . In thes and 925  and FAK- and 925 . Thus, tsal TEER  and FRNKsal TEER . Furthersal TEER , direct sal TEER . Confirmsal TEER . Further at Y658 . in vivo was provided by Schmidt et al. [A definitive proof for a requirement of endothelial FAK promoting endothelial barrier functiont et al. , by showt et al.  showed tt et al. . K545R Ft et al. . The datt et al. . It willAdherens junction proteins can bind to at least 12 distinct tyrosine phosphatases . OSrc homology-2 (SH2) domain-containing phosphatase 2  is a ubiquitously expressed phosphatase that is associated with multiple neoplastic malignancies, as well as three closely related inherited developmental disorders –182, theβ-catenin, and γ-catenin. This increase correlated with SHP2 phosphorylation and a loss of SHP2 in the VE-cadherin immunoprecipitates. Direct association of SHP2 with β-catenin was likely, since a far-Western blot assay demonstrated that a construct consisting of GST-tandem SHP2 SH2 domains bound to isolated β-catenin, but not VE-cadherin, p120, or γ-catenin from HUVEC lysates [β-catenin phosphorylation that lasted 15–30 minutes [β-catenin phosphorylation and the recovery of TEER. To prove that SHP2 mediated the recovery, it was shown not only that SHP2 immunoprecipitated from cells treated with thrombin was able to dephosphorylate β-catenin in vitro, but also that SHP2 knockdown prolonged β-catenin phosphorylation and thrombin-induced TEER loss [β-catenin as measured by IP and phosphotyrosine Western blot [ Pseudomonas aeruginosa [A first indication that SHP2 may regulate endothelial cell-cell contacts was provided by Ukropec et al. . In HUVE lysates . Consist minutes . ThrombiEER loss . AdditioEER loss . In vascEER loss . Proof tEER loss . In thesern blot . More reern blot . Furtherruginosa , suggest in vivo, since inhibition of FGF signaling in rats using adenoviral delivery of FGF traps destabilized the vasculature integrity and promoted vascular barrier loss [SHP2 also regulates the response to other vasoactive mediators. An early observation that VEGFR2 phosphorylation in response to VEGF-165 was much higher in HUVECs grown on vitronectin than in cells grown on collagen I  was attrier loss . Subsequier loss . FGFR1DNier loss .η) is a ubiquitously expressed phosphatase that was originally cloned from a HeLa cDNA library [ In vitro, Dep1 was found to bind directly to Src [β-catenin [β-catenin, and γ-catenin in lysates from HUVECs treated with pervanadate, but not control cells [ in vitro Dep1 was shown to dephosphorylate p120, in A431D cells Dep1 promoted an increase in the phosphorylation level of Y228 p120 after calcium addition. Importantly, the authors found that Dep1 expression also increased junctional VE-cadherin when expressed in HUVECs, although it remains unexplored whether the same p120-dependent mechanism is governing the action in HUVECs [Dep1 , in whiol cells . Coexpreol cells , a mutanol cells . In a caol cells . Even thn HUVECs . in vivo and to colocalize with VE-cadherin [ in vivo. A mechanism for the Dep1-mediated regulation of cell proliferation via the modulation of VEGFR2 signaling was proposed by Lampugnani et al. [β-catenin and its interaction with VE-cadherin. Expression of a catalytically inactive C/S Dep1 mutant or siRNA-mediated Dep1 knockdown restored VEGF-induced VEGFR2 phosphorylation and cell proliferation in confluent cells [γ, eNOS, and Erk1/2, but prevented VEGF-induced Akt activation [Dep1 was also shown to be expressed in the endotheliumcadherin , but itscadherin . Paradoxcadherin , suggestcadherin , thus coi et al. . Endothent cells , suggesttivation . Consisttivation , this wativation . In startivation . Reciprotivation . BAECs etivation .β) is an endothelial-specific transmembrane tyrosine phosphatase that was cloned from a bEnd5 cDNA library [γ-catenin [γ-catenin, suggesting that this phosphatase can enhance VE-cadherin/γ-catenin binding, but a mutant VE-cadherin in which all C-terminal tyrosine residues were replaced by phenylalanine also bound γ-catenin in the presence of VE-PTP, demonstrating that the effect of VE-PTP in γ-catenin binding was independent of the tyrosine phosphorylation level of VE-cadherin [β-catenin, and γ-catenin phosphorylation [ in vivo [β-induced inflammation of the cremaster muscle model, without affecting leukocyte attachment or rolling, and reduced LPS-induced increase of PMN in bronchoalveolar lavage (BAL) fluid. Rapalog injections also diminished VEGF and LPS-induced vascular leakage  [VE-PTP  . A role , resp.) . In this, resp.) .−/− allantois, demonstrating that Tie2 mediated this effect. Further supporting VE-PTP-Tie2 axis controlling vessel growth, daily injections of anti-VE-PTP antibodies for 7 days in young mice induced vessel enlargement in the tongue and Tie2 phosphorylation in lung lysates [ in vitro assay VE-PTP was able to directly dephosphorylate VEGFR2 [VE-PTP also plays a critical role in vascular development via its regulation of Tie2 and VEGFR2 signaling. VE-PTP null mice  or mice  lysates . VE-PTP  lysates . VE-PTP  lysates . This wa lysates . Further lysates . The same VEGFR2 . The mece VEGFR2 . This efe VEGFR2 .μ is a transmembrane receptor protein tyrosine phosphatase that was isolated using degenerated PCR primers from mouse brain cDNA based on its homology to other tyrosine phosphatases [μ localizes to cell-cell junctions in MvLu mink lung epithelial cells and coprecipitates with E-cadherin, β-catenin, and α-catenin [ In vitro, PTPμ was able to bind directly to the intracellular domain of E-cadherin but not to α-catenin or β-catenin. Pervanadate treatment did not prevent the coprecipitation of PTPμ with E-cadherin, suggesting that PTPμ can also associate with hyperphosphorylated cadherins [μ in WC5 neonatal rat cerebellar cells [μ association. The PTPμ binding site in E-cadherin was located to the C-terminal 38 amino acids, close to the β-catenin binding site [μ can sustain E-cadherin adhesion in LNCaP prostate carcinoma cells through a mechanism that involves scaffolding, but not catalytic activity [μ association is not restricted to E-cadherin, as PTPμ coprecipitated with E-cadherin, N-cadherin, and R-cadherin in rat lung extracts [μ coprecipitated with VE-cadherin, and in vitro GST pull-downs demonstrated a direct interaction between PTPμ and VE-cadherin [μ knockdown or expression of catalytically inactive constructs increased permeability to albumin. This effect may be due to regulation of VE-cadherin phosphorylation, as PTPμ overexpression in immortalized HMEC-1 cells reduced basal VE-cadherin tyrosine phosphorylation [μ in endothelial cells appears to be variable. PTPμ expression increased severalfold with increasing monolayer confluence in HUVECs [ In vivo, its expression may be restricted to arteries. Immunofluorescence studies showed that PTPμ expression was higher in arterioles and arteries than in veins of multiple rat tissues [μ-LacZ knock-in mice displayed β-galactosidase activity in arterioles and capillaries, but not in veins or in fenestrated endothelium [μ [μ differential expression as the sole explanation.PTPphatases . It contphatases . PTPμ lo-catenin . In vitradherins . Howeverar cells , suggesting site . Interesactivity . AJ/PTPμextracts  and promextracts . In humacadherin . This inrylation . Expressn HUVECs  and bovin HUVECs . In vivo tissues , and PTPothelium . This obothelium . Howeverelium [μ ) increaselium [μ  argues aβ-catenin. N-cadherin binding to PTP1B requires PTP1B tyrosine 152 phosphorylation [β-catenin dephosphorylation [μ [β-catenin binding site, although β-catenin and PTP1B do not appear to compete for N-cadherin binding [β-catenin, it is possible that PTP1B may affect junctional stability through the direct modulation of Src activity [αIIbβ3-integrin in platelets triggers PTP1B recruitment to a complex involving Src, Csk, and integrins, which leads to Csk dissociation and Src activation through dephosphorylation of tyrosine 530 [β3 integrin. Thus, not only can PTP1B be activated by tyrosine kinase signaling, but it can also promote tyrosine kinase activation. However, little is known about whether a PTP1B-Src axis is important in the endothelium. PTP1B was shown to bind and dephosphorylate VEGFR2 in vitro, and expression of wild-type PTP1B, but not a C/S catalytically inactive mutant, prevented VEGF-induced VEGFR2 phosphorylation and Erk, but not p38, in HUVECs [β-catenin, and VE-cadherin in rat lung microvascular endothelial cells as well as mouse lungs [β-catenin. More importantly, expression of an oxidation-resistant PTP1B mutant reduced LPS-induced lung edema [PTP1B is a ubiquitously expressed nonreceptor tyrosine phosphatase that holds the record of being the first tyrosine phosphatase purified and characterized –237. PTPrylation , 241. Inrylation , 239. Siation [μ , PTP1B/N binding . Besidesactivity –246. Forsine 530 . PTP1B in HUVECs . Conversn HUVECs . PTP1B cse lungs . LPS treng edema .This year marks the 30th anniversary of the first detection of phosphotyrosine at the cell-cell junctions . Since t in vivo with cadherin dephosphorylation of specific tyrosines [The ability of SFKs to phosphorylate VE-cadherin and the requirement for SFK activation downstream of multiple receptors, including VEGFR2 and ICAM-1, have been demonstrated. Nevertheless, it became clear that simply the observation of SFK activation cannot predict a loss of endothelial barrier function . The finyrosines , 91 and yrosines . These oyrosines , 250. Inyrosines , a proceβ-catenin, α-catenin, and the actin cytoskeleton. Additionally, it is possible that some phosphorylation events in VE-cadherin may be a consequence, and not a cause, of catenin dissociation. The adherens junctions associate with multiple tyrosine kinases [β-catenin can bind and recruit them to the junction or, alternatively, compete for a binding site and displace kinases and phosphatases from binding VE-cadherin, with the overall effect of modulating the levels of junctional phosphotyrosine (A potential mechanism that stands out for its simplicity and logic is the regulation of the association of VE-cadherin to catenins by differential phosphorylation events. The well-known role of p120 in preventing VE-cadherin endocytosis , 129 and kinases , 172 and kinases , 213, antyrosine . The exa


Aims: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), β-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. Methods: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, β-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. Results: AMACR and β-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and β-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. Conclusions: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of β-catenin. Chordomas are locally aggressive malignant tumours of notochordal origin that typically arise in bones of the base of skull, the vertebral bodies and the sacrococcygeal bone. They account for 1–4% of all primary malignant bone tumours343536The pathological differential diagnosis of chordomas includes chondrosarcoma, metastatic carcinoma, myxopapillary ependymoma and chordoid meningiomaChondrosarcoma is the third most common malignant tumour of the bone (20% of the cases) after myeloma and osteosarcoma. The bones of the pelvis are the most common skeletal site of chondrosarcoma but rarely, chondrosarcoma may involve the spine and the base of the skull. Likewise chordomas, chondrosarcomas are slightly more common in men than in women and peak at the same age rangeHistologically, chondrosarcomas invade the bone, creating irregularly shaped lobules with some fibrous bands. The chondrocytes are surrounded by abundant cartilaginous/myxoid matrix. The morphological features vary with tumour grade and more aggressive tumours have higher degrees of cellularity and nuclear pleomorphism2339Therefore, the distinction between chrodomas and chondrosarcomas has important clinical implications. Since the two entities share some clinical and morphological features, the differentiation between them at times may be difficult. In fact, gene expression analysis has found that of all cartilaginous neoplasms chordomas are most closely related to chondrosarcomas551314E-cadherin and β-catenin belong to the Wnt signaling pathway and react with each other171819In view of the reported association of chordomas with β-catenin staining and β-catenin with AMACR expression, we hypothesized that chordomas are associated with overexpression of AMACR. To test our hypothesis we have assessed by IHC stains the expression of E-cadherin, β-catenin and AMACR in a cohort of chordomas and compared it to a set of chondrosarcomas. Next we evaluated mRNA level of AMACR. To conclude, we compared the capacity of these three IHC markers to differentiate chordomas from chondrosarcomas.The study was approved by the institutional Helsinki committee. We have identified in the archive of the Department of Pathology at the Sheba Medical Center 18 chordomas, 19 chondrosarcomas and 10 cases of mature cartilage tissue. All samples were resected from 1994–2014. The diagnosis of all cases was revised by the study pathologist (SAR). Guided by routine hematoxylin & eosin (HE) stained slides, representative formalin-fixed paraffin embedded (FFPE) block from each case was chosen for IHC study.FFPE blocks were sectioned at 4 μm and a positive control was added to each slide. All IHC staining was fully calibrated on a Benchmark XT staining module . Briefly, after sections were dewaxed and rehydrated, a CC1 Standard Benchmark XT pretreatment for antigen retrieval  was selected for all immunostains: AMACR , β-catenin, , E-cadherin,  and brachyury . Detection of AMACR, β-catenin and E-cadherin was performed with iView DAB Detection Kit  and of brachyury with ultraView DAB Detection Kit . Slides were counterstained with hematoxylin  on an automated stainer and dehydrated in ethanol solutions  for one minute each. Before cover-slipping, sections were cleared in xylene for 2 minutes and mounted with Entellan .All slides were examined under Olympus BX50 microscope. IHC staining was scored by the study pathologist (SAR). The cut-off for positivity was ≥10% of cells with positive staining. AMACR showed granular cytoplasmic staining, E-Cadherin had membranous and cytoplasmic staining and β-catenin was scored for cytoplasmic staining. The intensity of staining was scored as well but was not included due to its lack of statistical significance. Microscopic images were taken using Olympus DP71 camera and Cell^B software.−ΔΔCT methodT for each gene was calculated from the triplicate readings. Next, for each sample the geometric mean of the averaged CT of the housekeeping genes was calculated and served as the endogenous reference. Finally, 2−ΔΔCT method was applied using the readings of the prostatic adenocarcinoma sample for calibration.Total RNA was extracted from FFPE tumour samples of 8 chordomas and 3 chondrosarcomas with relatively large tumour tissue required for the procedure. Total RNA extracted from FFPE tumour sample of prostatic adenocarcinoma was used as a reference  sample. The RNA was extracted using the RNeasy FFPE kit  according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using the qScript cDNA Synthesis kit  and includes the use of a mixture of random and Oligo (dT) primers and recombinant Moloney-murine leukemia virus RT as described in the manufacturer’s protocol. Quantitative RT-PCR amplification was performed with Platinum® SYBR® Green qPCR SuperMix  in a 16 μl reaction volume containing 2 μl of cDNA products as a template. The expression of three housekeeping genes GAPDH, β-actin and HPRT was used for normalization. Primers were designed using the primer express software (GAPDH) and the primer 3 software (β-actin and HPRT). All of the primer pairs designed were verified for specificity by comparing to the human RefSeq database of mRNAs (by using Blast search for short nearly exact matches). The primer sequences used were as follow: GAPDH , β-actin , HPRT , AMACR Statistical analysis was carried out on R Statistical Software, using the Fisher’s exact test, one-sided for evaluation of each staining, exact conditional test, two-sided for comparison of differentiating capacity of AMACR to β-catenin, unpaired two-sided Kruskal–Wallis test for age assessment and Fisher’s exact test, two-sided for gender study. Statistical significance was defined in general as P < 0.05, and the alpha level was adjusted for multiple comparisons using the Bonferroni correction for evaluation of each staining and for comparison of differentiating capacity of AMACR to β-catenin: alpha = 0.05/13 = 0.0038.Std 13.98). The chordomas were located in the chest wall (1), cranium (6) and along the spine (11). Chondrosarcoma patients consisted of 8 females and 11 males with a mean age at surgery of 45.7 years . The chondrosarcomas were located in the chest wall (2), femur (4), arm (6), pelvis (4) and spine (3). Among the mature cartilage patients were 8 females and 2 males with a mean age at surgery of 53.7 years . Mature cartilage samples came from a variety of anatomic sites (5) and from femoral head (5). There was significant statistical dominance of age of the chordoma group over the chondrosarcoma group (p = 0.011), but no other significant statistical dominance of age between the study groups . As for gender prevalence, both the chordoma and chondrosarcoma groups contained more males than females with no significant difference between the two groups . In comparison with mature cartilage there is statistically significant difference only for chordoma .The characteristics of the study cohort are summarized in Brachyury immunostain confirmed the diagnosis of all cases in our cohort as it stained positively all the chordomas (aside from one case in which tissue was exhausted) and none of the chondrosarcoma or mature cartilage samples.Thereafter we compared the percentage of cases that were positively stained by AMACR, β-catenin and E-cadherin in the three study groups. p = 3.839e-05, odds ratio = 26.2) and to mature cartilage . However, no significant difference was found for AMACR between the percentage of positively stained cases in chondrosarcoma and mature cartilage (p = 0.16).AMACR stained positively 88.9% of the chordomas, only 21.1% of the chondrosarcomas and none of the mature cartilage cases. The percentage of positively stained cases was significantly higher in chordoma compared to chondrosarcoma  and to mature cartilage . No significant difference was found for β-catenin between the percentage of positively stained cases in chondrosarcoma and mature cartilage (p = 0.53).β-catenin staining showed in our cohort only cytoplasmic staining pattern. No sample has shown nuclear staining. β-catenin stained positively 94.1% of the chordomas, only 10.5% of the chondrosarcomas and none of the mature cartilage cases. Likewise AMACR, the percentage of β-catenin positively stained cases was significantly higher in chordoma compared to chondrosarcoma , this difference did not reach the minimal level required (0.0038) for statistical significance following the Bonferroni correction for multiple comparisons. No difference in E-cadherin staining was found between chordoma and mature cartilage (p = 0.08) or between chondrosarcoma and mature cartilage (p = 0.77).E-cadherin, stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Although the percentage of E-cadherin positively stained cases was higher in chordoma compared to chondrosarcoma (p = 0.74) and chordomas from mature cartilage (p = 1).Our results did not show significant difference between the ability of AMACR and β-catenin staining to differentiate chordomas from chondrosarcomas .The characteristics of our cohort are similar to the described in the literature for chordomas and chondrosarcomas. The continuous search for IHC markers that would allow a better distinction between chordomas and chondrosarcomas is still relevant due to the existing overlap between the IHC profiles of the available markers. During the last decade few differentiating IHC markers have been suggested. In view of the epithelioid features that chordoma cells display, different adhesion molecules have been studied as IHC markers of chordomas41314β-catenin has a role in two pathways involved in tumourigenesis. First, it affects cell adhesion and suppresses tumour invasion through its interaction with E-cadherin at the cell surface that causes recruitment of α-catenin, which in turn binds the intracellular actin cytoskeleton. Second, it is a key mediator in the Wnt signaling pathway which regulates cell proliferation and differentiation. A multi-protein complex that includes adenomatous polyposis coli (APC) and glycogen synthase kinase-3 β (GSK3 β) promotes the degradation of β-catenin thus controls the levels of free β-catenin in cells. Activation of Wnt pathway leads to an inactivation of GSK3 β, which allows β-catenin levels to rise in the cytoplasm, and also to translocate to the nucleus where it activates Wnt-related transcription factors and genes3et al.AMACR is known to be overexpressed in variety of tumours including prostatic adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma and colorectal cancers17181920252627In our current study we have shown for the first time that chordomas express AMACR both at the protein as well as the mRNA level. Moreover, we have found that AMACR may serve as IHC marker of chordoma, assisting in the differential diagnosis with chondrosarcoma. AMACR differentiating utility appeared to be comparable with that of β-catenin.Our attempts to study AMACR at the mRNA level yielded confirmation of its presence. Yet, in view of the technical problems that we have encountered, leading to narrowing of the cohort, a further study, preferably using fresh frozen tissue is recommended.AMACR should be used for differential diagnosis of chordomas from chondrosarcomas in combination with the classical stains EMA and CKs. This panel will cover the reported possibility of negative expression in chordomas and positive expression in chondrosarcoma. Likewise CKs, AMACR may be focal and therefore the use of AMACR as part of a panel of stains is advised especially when handling small biopsies.Brachyury is highly specific marker for chordomas, therefore it is recommended for the work out of differential diagnosis with chondrosarcomas. Accordingly, we used brachyury to confirm the diagnosis of all samples in our cohort. However, since the sole clinical indication for use of brachyury is to identify chordomas, many laboratories choose not to include it in their routine repository due to economical considerations. AMACR, on the other hand, that has varied diagnostic uses is available at most laboratories. Thus its higher accessibility compensates for the lower specificity relative to brachyury.Last but not least, the differential diagnosis of chordoma includes metastatic carcinoma, aside from chondrosarcoma. In cases suspected for chordoma, where the possibility of metastatic carcinoma of prostatic, hepatic, renal or colorectal origin is considered, AMACR and β-catenin IHC stains cannot assist the differential diagnosis since all of these tumours express both markers. In these cases the differential diagnosis requires the use of other markers such as S-100 and other tumour specific proteins according to the presumed origin . Moreover, brachyury has been recently found to be expressed in a number of carcinomas including hepatocellular carcinoma, lung, colon and breast carcinomas293031In conclusion, our study is the first to show that AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. Thus, AMACR may serve as IHC marker of chordoma in combination with the classical stains EMA and CKs, as it shows differentiating ability comparable to that of β-catenin. Nonetheless, more studies are needed to substantiate our results and to identify the specific role of AMACR in chordoma.How to cite this article: Aviel-Ronen, S. et al. α-methylacyl-CoA racemase (AMACR) expression in chordomas differentiates them from chondrosarcomas. Sci. Rep. 6, 21277; doi: 10.1038/srep21277 (2016).


Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg) exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg). Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history of safe human use, our discovery of fluspirilene as a potential anti-HCC drug may present an immediately applicable clinical therapy.Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC’s resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK) pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested  Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Only 30% to 40% of the HCC patients are eligible for curative treatments, which include surgical resection as the first option, liver transplantation and percutaneous ablation. However, there is a high frequency of tumor recurrence after surgical resection, and most HCCs seem resistant to conventional chemotherapy and radiotherapy. Therefore the development of novel therapies against HCC is highly demanded.The cause of HCC involves multiple pathways. The cyclin-dependent kinase (CDK) pathways as important therapeutic targets for cancer treatment have been well established. CDKs are enzymes implicated in cell replication, and their role in tumor growth has long made them into attractive drug targets. But early industrial attempts at inhibiting CDKs to restore cell growth to normal have encountered toxicity issues. First-generation CDK inhibitors were non-specific, inhibiting many different CDKs , and resulting in the type of toxicities and muted efficacy seen with older chemotherapies.Cyclin-dependent kinase 2 (CDK2) is one of the serine/threonine protein kinases. It plays a pivotal role in regulating the cell cycle transition from G1 to S phase, and thus in controlling cell proliferation. Hence, CDK2 inhibitors are potentially effective anti-cancer agents. Although a number of CDK2 inhibitors have been described in the literature  and olomin silico approach of structure-based virtual screening and ensemble docking to repurpose approved drugs for the treatment of cancers that involve CDK2 regulation, with a major focus on human hepatocellular carcinoma (HCC). We tested nine computationally favoured compounds in vitro in HCC cell lines HepG2 and Huh7, and successfully identified the anti-psychotic drug fluspirilene as a potential CDK2 inhibitor. We then performed in vivo experiments in nude mice xenografted with Huh7 cells, and showed that fluspirilene exhibited strong anti-tumor activity comparable to that of the leading cancer drug 5-fluorouracil, further establishing fluspirilene as a candidate anti-cancer drug. We also showed that the cocktail treatment with both fluspirilene and 5-fluorouracil could produce synergistic therapeutic effect. Finally, we analyzed the predicted binding conformation of fluspirilene and revealed the critical intermolecular interactions that possibly govern fluspirilene binding to CDK2.In this study, we used our free and open-source protein-ligand docking software idock , 6 to scThis study was approved by the laboratory animal ethics committee of Kunming Medical University.There are as many as 346 solved X-ray crystallographic structures of CDK2 from the PDB (Protein Data Bank) , 8 with To test the redocking accuracy of idock, the co-crystallized ligands were also manually extracted from their corresponding complexes and converted from PDB format to PDBQT format using the prepare_ligand4.py script of AutoDockTools . These lThe structures of FDA-approved drugs were obtained from the dbap and fda catalogs of the ZINC database , 11, wheOur free and open-source docking software idock v2.1.2  was thenhttp://www.chemicalbook.com/ and subsequently validated in vitro and in vivo.After docking, idock outputted a maximum number of nine predicted conformations for each input compound. The docked conformation with the best idock score was selected because it was previously shown to be the most likely one closest to the crystal conformation with a redocking success rate of more than 50% on three different benchmarks . The 4,9The selected chemicals and the leading cancer drug 5-fluorouracil were purchased from Sigma-Aldrich, USA. Anti-cyclin D, B1, E, CDK2, Rb, pho-CDK2 (Thr160), pho-Rb (Ser795) and GAPDH were obtained from Cell Signaling Technology, Inc., Danvers, Massachusetts, USA.2 and 95% humidified air.Hepatoma cell lines HepG2 and Huh7 were obtained from the American Type Culture Collection, Manassas, Virginia, USA. These cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS)  at 37°C in 5% COμM, and incubated for 24, 48, or 72 hours.Cells were plated in 96-, 24-, or 6-well plates with 0.125% FBS medium for 24 hours and then treated with 10% FBS medium containing the testing compounds at various concentrations of 1, 3, 10, 30 3 cells/well in 96 well plates and incubated with 0.5mg/ml 3--2,5-diphenyl-tetrazolium bromide for 4 hours. The medium was then discarded and 200 μl of formazan in dimethylsulphoxide (DMSO) was added. The absorbance was measured at 570 nm according to the standard protocol. The IC50 values were calculated by GraphPad Prism 5.Cells were plated at an initial density of 9 × 104) were seeded in 24-well plates in RPMI 1640 medium containing 0.125% FBS, and cultured for 24 hours. The cells were incubated in medium containing 10% FBS and various doses of fluspirilene  for 12, 24, 36 hours at 37°C, then fixed in ice-cold 70% ethanol and stained with a Coulter DNA-Prep Reagents kit . Cellular DNA content of 1 × 104 cells from each sample was determined with the use of an EPICS ALTRA flow cytometer (Beckman Coulter). Cell cycle phase distribution was analyzed with the ModFit LT 2.0 software . All results were obtained from two separate experiments, each of which was done in triplicate.HepG2 or Huh7 cells  (Life Technologies). Briefly, cells were trypsinized with 0.25% trypsin in the absence of ethylenediamine tetraacetic acid (EDTA). The cells were washed with PBS twice and resuspended in 500 μl of binding buffer at a concentration of 2 × 105 − 106 cells/ml. Two microliters of annexin V-EGFP and 5 μl of PI were added to the suspension followed by 5 to 15 minutes of incubation in the dark. The cells were then analyzed using flow cytometry .HepG2 and Huh7 cells were plated at 24-well plates with 0.125% FBS medium for 24 hours and then with 10% FBS medium containing fluspirilene at concentrations 3, 10 and 30 μM. Cells were harvested after 6 hours of incubation. Cells were lysed with RIPA buffer containing 1 mM PMSF and protease inhibitor cocktail at 4°C for 30 minutes. After centrifugation at 13,000 rpm for 15 minutes, the supernatants were recovered and the protein concentration was measured by BCA Protein Assay Kit (Thermo). Equal amounts of cell lysates were resolved in 10% SDS-PAGE and transferred onto nitrocellulose membranes (Sigma). After blocking, the membranes were incubated sequentially with the appropriate diluted primary and secondary antibodies. Proteins were detected by the enhanced chemiluminescence detection system . To ensure equal loading of the samples, the membranes were re-probed with an anti-GAPDH antibody .HepG2 and Huh7 cells were plated at 6-well plates with 0.125% FBS medium for 24 hours and then with 10% FBS medium containing fluspirilene at concentration 3, 10, 30 6/0.2ml PBS Huh7 cells were injected subcutaneously into the right flank of the mice. Tumor size was measured every day. Three weeks after inoculation when the tumors grew to a volume of 80 to 100 m3, the mice were randomly divided into groups of 5 mice per group, and fed by oral gavage with 0.5% CMC-NaCl containing fluspirilene (15mg/kg) and by intraperitoneal injection of 5-fluorouracil (10mg/kg) daily for 21 days. Carcinoma volumes were measured every 3 to 4 days after tumor appearance. Tumor volume was calculated by the formula V = ab2/2, where a is the longest axis and b is shortest axis. The mice were then sacrificed by cervical dislocation.Female BALB/C nude mice, 4 to 5 weeks old from Vital River Laboratory Technology Co. Ltd, Peking, China, were kept under specific pathogen-free conditions. For the xenografted tumor growth assay, 1 × 10The results were obtained from at least three different experiments and expressed as mean ± SD. Statistical analysis was performed by Student’s t test and differences were considered to be statistically significant if p < 0.05. Statistically significant results are marked with the * symbol in the figures.For the 44 CDK2 complexes, the co-crystallized ligands were docked against their corresponding CDK2 structure to see if idock could predict binding poses as conformationally close to the co-crystallized pose as possible. Such redocking accuracy was quantified by the root mean square deviation (RMSD) of the predicted poses from the co-crystallized pose. For each of the 44 complexes, idock predicted nine poses, hence nine RMSD values. The RMSD values of the nine poses in the 44 redocking trials are provided in http://istar.cse.cuhk.edu.hk/idock/iview/?1GZ8-dbap and http://istar.cse.cuhk.edu.hk/idock/iview/?1GZ8-fda.Totally 4,914 FDA-approved drugs were docked and ranked according to their average predicted binding affinity across 44 X-ray crystal structures of CDK2. Their predicted free energy values are provided in 50, i.e. 4.017 μM for HepG2 and 3.468 μM for Huh7. Fluspirilene exhibited the highest cytotoxicity compared to the control with statistical significance (p < 0.05). Such inhibition effect was dose- and time-dependent. Marked inhibition was observed at 10 μM and 30 μM, but no significant effect was observed at concentrations below 3 μM.We first evaluated the anti-cancer effect of the nine compounds by MTT assay . All theμM for 6, 12, 24 hours on cell cycle profile in HepG2 and Huh7 cells by flow cytometry . The scatter plots of PI against annexin V are provided in A drug that can only induce cell cycle arrest is not good enough because the tumors will easy re-grow after withdrawing the drug. Therefore we also investigated whether fluspirilene could induce cell apoptosis . FluspirWe investigated the effect of fluspirilene on the expressions of critical proteins involved in G1-to-S transition in HepG2 and Huh7 cells by western blotting . FluspirIt is also observed that fluspirilene treatment majorly decreased the expression rather than phosphorylation of CDK2. We propose that such differences are reasonable in the sense that after binding, CDK2-fluspirilene complexes may directly undergo degradation, therefore resulting in higher reduction of CDK2 expression than phosphorylation. In addition, fluspirilene may bind to CDK2 with a high affinity, but after CDK2 is phosphorylated, the binding of fluspirilene may be impaired and the binding affinity could thus be weakened.in vivo in BALB/C nude mice subcutaneously injected with Huh7 cells butoxypurine (codenamed MBP for short), which is an O(6)-substituted guanine derivative, is known to establish four hydrogen bonds with Lys33, Glu81 and Leu83  was highlighted in several recent publications [in silico molecular modeling and docking studies along with drug formulation and in vivo anti-inflammatory inspection; [1 as an inhibitor of NEDD8-activating enzyme (NAE) in cell-free and cell-based systems with high selectivity.The powerful synergy of ications . To nameications  rediscovpection;  attempteIn addition to SBVS, ligand-based virtual screening (LBVS) also finds its successful applications in repurposing.  used UltEncouraged by these successful stories, in this study we adopted the repurposing strategy, and utilized the computational methodology of SBVS by protein-ligand docking to shortlist candidates from FDA-approved small molecule drugs. Specifically, we used our fast docking program idock , 6 in cohttp://istar.cse.cuhk.edu.hk/idock, where there are as many as over 23 million purchasable small molecule compounds ready for docking against any protein supplied by the user. As of 1 May 2015, there have been more than 1000 docking job submissions by users from 98 countries. Both idock [To facilitate the use of idock, its input arguments and output results were purposely designed to be similar to those of AutoDock Vina, therefore existing users can easily transit to idock and benefit from considerable speedup in SBVS performance. Moreover, to promote prospective SBVS by idock, a web server called istar  was deveth idock  would hoRegarding the structural data in use, so far there are as many as 346 solved X-ray crystal structures of CDK2 with a UniProt ID of P24941 . To acco50 was less than 10 μmol/L as determined by MTT assay. Fluspirilene is currently used for the therapy of chronic schizophrenia. It was recently identified as a potential p53-MDM2 inhibitor and its anti-cancer effect in vitro was reported [in vitro in HCC cell lines, nor in vivo in nude mice, and its role as a potential CDK2 inhibitor remained unknown until now.After ensemble docking experiments with idock , 6 folloreported , but wasin vivo in nude mice xenografted with hepatoma Huh7 cells. Most importantly, the combination of effective dose of fluspirilene and 5-fluorouracil produced even higher therapeutic effect, indicating that fluspirilene may work through a different mechanism than 5-fluorouracil, which further indicates that fluspirilene could be combined with other chemotherapy drugs to achieve synergistic therapeutic effect. Our finding is particularly inspiring in that fluspirilene could be the first CDK2 inhibitor used for HCC therapy.In this study, we reported for the first time that fluspirilene is a potential CDK2 inhibitor, and demonstrated for the first time that oral administration of fluspirilene (15 mg/kg) exhibited significant and strong anti-cancer efficacy comparable to the leading cancer drug 5-fluorouracil (10 mg/kg) in situ hepatocellular carcinoma models with HCM-Y89 cell by intravenous injection was 48.14% at 20 day of post-treatment [No obvious toxicity was previously reported by intraperitoneal injection of fluspirilene (8 mg/kg) in male wistar rats . In thisreatment . In thisin vitro and in vivo in HCC cells only, we believe fluspirilene may also exhibit anti-cancer effect in other types of cancers.Last but not the least, although we tested fluspirilene This study presents a successful prospective application of idock , 6 in idS1 TableThe table comprises the PDB ID, resolution, chain and positions.(CSV)Click here for additional data file.S1 FigThe molecular surface of CDK2 is colored by secondary structure, with an opacity of 0.9 to show the underlying secondary structure in cylinder & plate representation. ATP is rendered in stick representation colored by atom type. Waters are shown as red dots and metal ions are shown as green dots. This figure was created by iview .(PNG)Click here for additional data file.S2 TableThe table comprises the PDB ID, the pose number, the idock score and RMSD.(CSV)Click here for additional data file.S3 TableThe table comprises the ZINC ID, the catalog, the average score, standard deviation and individual scores for the 44 selected CDK2 structures, and the molecular properties.(CSV)Click here for additional data file.S4 TableThe table comprises the ZINC ID, the average score, scientific name, clinical uses and references.(PDF)Click here for additional data file.S2 FigCDK2 residues are rendered as lines colored by atom type. MBP is rendered as sticks colored by atom type. The interacting atoms and residues are labeled. The cyan dashed lines represent hydrogen bonds. This figure was created by iview .(PNG)Click here for additional data file.S3 FigCDK2 is rendered as molecular surface colored by atom type, with an opacity of 0.9 to show the underlying atoms. Fluspirilene is rendered as sticks colored by atom type. This figure was created by iview .(PNG)Click here for additional data file.S4 FigCDK2 is rendered as molecular surface colored by atom type, with an opacity of 0.9 to show the underlying atoms. MBP is rendered as sticks colored by atom type. This figure was created by iview .(PNG)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.S6 Fig(JPG)Click here for additional data file.


Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT) of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB) and of primary embryo fibroblasts (Mefs) was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF) of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP) show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and β-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.Protein arginine methyl transferase 5 (Prmt5) regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which  Adipose tissue has various regulatory functions in the metabolism of animals and acts both as a fat reservoir and an endocrine/paracrine/autocrine organ that can expand throughout the entire lifespan. This functional plasticity can lead to pre-adipocyte hyperplasia and adipocyte hypertrophy. Fat tissue includes many different cellular components, including preadipocytes, multipotent stem cells (MSC) and mature adipocytes .Adipogenesis is a multi-step process during which the increase in adipocyte number is triggered by various extra- and intra-cellular signalling factors that induce MSC conversion into preadipocytes . This coIn association with protein complexes involved in different phases of transcription, Protein arginine methyl transferase 5 (Prmt5) was implicated in myogenic, adipogenic and glial cell differentiation . ConsistCopr5 was genetically invalidated and show that adipogenic conversion was delayed in vitro both in EBs and Mefs derived from these mice compared to control cells. In addition, the retroperitoneal WAT of Copr5 KO (KO) mice was slightly reduced and contained larger adipocytes compared to control mice. Finally, we show that the expression of Dlk-1 was upregulated in KO cells and coincides with an altered recruitment of Prmt5 and β-catenin to the Dlk-1 promoter. Altogether, our data highlight unsuspected functions of Copr5 in the modulation of adipogenic differentiation, notably through an impact on the Wnt/β-catenin-dependent regulation of the Dlk-1 promoter.In this work, we generated mice in which Copr5 gene was genetically invalidated by homologous recombination . In contrast to Prmt5 loss of function, which is early embryonic-lethal due to loss of pluripotent cells . Altogether, these data indicate that Copr5 is required for an efficient adipogenic conversion of cells in culture. Although the mRNA level of Copr5 did not vary significantly during fat tissue development  . Surprisingly, a transient ectopic re-expression of Copr5 in KO cells failed to rescue their capacity to differentiate . These results suggest that Copr5 deficiency had impacted on very early and irreversible events required for the adipogenic conversion of Mefs.We generated a mouse model in which the nt cells , Copr5 Kipocytes , lipid dipocytes . Moreoveo WT EBs . O Red Oo WT EBs , as wellig. S2A) , it was and Klf5 . As expesupplementary material Table S1). Notably, among the 538 genes that were significantly deregulated  in KO cells, 34 were bona fide Wnt/β-catenin target genes  . Biochemical fractionation showed that KO Mefs contained higher amounts of the activated form of β-catenin in their nucleus than WT cells , a difference that was lessened upon treatment with either LiCl or C59, two chemicals known to activate and inhibit the Wnt pathway, respectively . Consistently, reporter assays confirmed that TCF/β-catenin transcriptional activity was increased in KO cells . Within this list, we noticed the presence of Dlk-1, a gene encoding a key regulator of adipose tissue homeostasis in vivo whose expression in WAT is linked to inhibition of adipocyte differentiation . Interestingly, we found that the recruitment of Brg-1, a chromatin remodeller that can be recruited by β-catenin to TCF target gene promoters and able to interact with Prmt5 . To which extent proteins that are able to antagonise β-catenin/TCF activity might be responsible of this reduced binding of β-catenin at the Dlk-1 promoter in KO cells still remains. We next assessed whether a shRNA-mediated depletion of Dlk-1 could restore the capacity of these cells to differentiate. Because they differentiated poorly once infected with shRNAs, we used Copr5-depleted F442A cells. Although a reduction of Dlk-1 level was obtained in these cells, this reduction was still unable to fully restore a capacity of differentiation , as expected  to the medium. ES cells and EBs were cultivated as described previously .Adipose tissue was dissected, washed in PBS with a 2% penicillin/streptomycin/gentamycin mixture, minced, and incubated in DMEM supplemented with 10 mg/ml BSA, 0.35% type II collagenase (SIGMA) at 37°C with shaking for 30 min. Cell suspensions were obtained after filtration through 100 µm cell strainers, centrifuged. The remaining pellet was resuspended, filtered through 40 µm cell strainers and centrifuged to recover the VSF.In vivo labelling was performed by intraperitoneal injection of either BrdU at a concentration of 50 µg BrdU/g body weight or PBS included as negative controls in 7-week-old animals that were sacrificed seven days later for VSF isolation. VSF was processed using the BrdU FITC kit, as recommended by BD Pharmingen.Sections of paraffin-embedded adipose tissue were stained with haematoxylin/eosin. Quantification was performed from images within a 500×500 µm measurement frame using ImageJ. Three independent measurements were performed in both WT and KO mice (n = 3).supplementary material Table S2.RNA isolation and RT-qPCR were performed as described . The seqsupplementary material Table S2.Anti-Prmt5 and -β-catenin (S33/37/41) antibodies  were used for ChIP, as described . SequencAnti-Prmt5 (Millipore), -β-catenin , Histone H3 (Millipore), -Dlk-1 (Abcam) and -Tubulin antibodies were used.Z ratio and the adjusted Zp value was >2 and 0.05, respectively.Microarray analysis was performed using total RNA isolated from either Copr5 KO or WT Mefs from male embryos (n = 3), hybridised onto a GeneChip® Mouse Gene 2.0 ST Array and analysed for differentially expressed genes  that were considered significant when the


Gastro-intestinal neuroendocrine tumors (GI-NETs) are rare neoplasms, frequently metastatic, raising difficult clinical and therapeutic challenges due to a poor knowledge of their biology. As neuroendocrine cells express both epithelial and neural cell markers, we studied the possible involvement in GI-NETs of axon guidance molecules, which have been shown to decrease tumor cell proliferation and metastatic dissemination in several tumor types. We focused on the role of Semaphorin 3F (SEMA3F) in ileal NETs, one of the most frequent subtypes of GI-NETs.in vitro. In two different xenograft mouse models, SEMA3F-overexpressing cells exhibited a reduced ability to form tumors and a hampered liver dissemination potential in vivo. This resulted, at least in part, from the inhibition of mTOR and MAPK signaling pathways.SEMA3F expression was detected in normal neuroendocrine cells but was lost in most of human primary tumors and all their metastases. SEMA3F loss of expression was associated with promoter gene methylation. After increasing endogenous SEMA3F levels through stable transfection, enteroendocrine cell lines STC-1 and GluTag showed a reduced proliferation rate This study demonstrates an anti-tumoral role of SEMA3F in ileal NETs. We thus suggest that SEMA3F and/or its cellular signaling pathway could represent a target for ileal NET therapy. Gastro-intestinal neuroendocrine tumors (GI-NETs) are uncommon neoplasms, but their incidence and prevalence are steadily increasing since the past 30 years . NETs arThis is the case for several “axon guidance” molecules which have been shown to be constitutively expressed by normal digestive neuroendocrine cells, along with their receptors –4. The pin vitro and in vivo approaches, that SEMA3F expression decreases with tumor progression in human ileal NETs and that this molecule is a negative regulator of neuroendocrine cell proliferation.The present work is focused on the semaphorin family of axon guidance molecules. Among semaphorins, class 3 molecules are secreted and might therefore be implied in paracrine or autocrine pathways . SecreteIn human tissue samples, immunohistochemical staining detected SEMA3F expression in the normal intestinal tissue adjacent to the tumor. The expression was restricted to a few cells scattered within the epithelial layer and to ganglion cells in the myenteric plexus. The endocrine nature of normal SEMA3F-positive epithelial cells was confirmed by their co-expression of chromogranin A (CgA), a specific peptidergic endocrine marker Figure . The SEMAmong the 24 primary tumors represented in TMAs, only one showed positive immunoreactivity for SEMA3F. All metastastic deposits represented in the second TMA were negative.P < 0.005)  Figure .P < 0.01) ; the number of Ki67 positive cells significantly increased with tumor stage (P < 0.01)  Figure . Further) Figure . These rSEMA3F promoter in a subset of 18 ileal NETs from 6 patients with multifocal lesions, including 5 T1, 8 T2 and 5 T3 tumors. An immunohistochemical staining of SEMA3F was performed on control tissue sections of the same tumor. All T1 tumors were unmethylated and positive for SEMA3F. Among the 8 T2 tumors analyzed, 3 were negative for SEMA3F expression but none was methylated. Among T3 tumors, the only 2 tumors positive for SEMA3F were not methylated; 2 out of the 3 SEMA3F-negative cases showed gene promoter methylation . SEMA3F expression in stable transfected clones was checked by immunoblot using MycTag antibody    Figure . STC-S3F) Figure .p-HH3 expression. Cell cycle analysis revealed a lengthened G1 phase in STC-1 and GluTag clones overexpressing SEMA3F (P < 0.05)  (P < 0.005)  Figure . Further) Figure . No chancontrols  indicatiin vivo studies because the experimental model based on STC-1 has been extensively characterized in our laboratory [2 clone exhibited a reduced weight loss compared to animals grafted with the STC-AP clone . This suggests a decrease in liver colonization and/or engraftment   Figure . This siA mouse model of intracaecal xenograft was then used because it closely recalls the clinical features and natural history of human GI-NETS, particularly the metastatic dissemination to the liver .2 clones were subcutaneously injected into nude mice. Subcutaneous STC-AP tumors reached an average volume of 75.89 ± 25.16 mm3 at 22 days while the volume of STC-S3F2 tumors was only 8.75 ± 4.75 mm3 at this stage. STC-S3F2 tumors reached a volume of 52.56 ± 15.47 mm3 at day 32   Figure .2/mm2 in controls to 4.80 ± 0.76 μm2/mm2 tumor tissue in STC-S3F2 intrahepatic nodules .Previous studies have shown that SEMA3F might affect several signaling pathways . To testo Figure . The sams Figure . Furthers Figure  whereas Among class 3 semaphorins, SEMA3F has been studied in several cancer types. The first evidence for a tumor suppressor effect came from lung cancer, in which the region of chromosome 3p21 containing SEMA3F coding gene are commonly deleted –14. SubsMost patients with ileal NETs present at diagnosis with locally advanced and often metastatic tumors. Therefore, to get further information about the relation between SEMA3F expression and tumor progression, we took advantage of a large series of multifocal ileal NETs. This unique setting allows to correlate SEMA3F expression with local invasion because of the coexistence in the same patient of tumors at different stages of local invasion . We obseSEMA3F gene promoter, suggested to be a potentially important regulator of SEMA3F gene expression [We then addressed the mechanisms responsible for SEMA3F loss of expression in neuroendocrine tumor cells. We paid attention to the methylation of pression . In a siin vitro and in vivo models. We first aimed to determine the expression profiles of SEMA3F and its preferential receptor NRP-2 in several neuroendocrine tumor cell lines. The highly proliferative STC-1 and GluTag cell lines exhibited very low SEMA3F and high neuropilin-2 expression, while the low proliferative INS-1E cell line, expressed strongly SEMA3F but faintly neuropilin-2. Interestingly, these cellular models retain the apparent correlation between SEMA3F loss of expression and high proliferative capacities.To further explore the functional consequences of SEMA3F loss of expression in neoplastic neuroendocrine cells, we turned to experimental in vitro and in vivo. In vitro, both STC-1 and GluTag cells showed a reduced proliferation rate when endogenous SEMA3F expression was restored through stable transfection. This decrease in proliferative activity seemed to be due to a lengthening of G1 phase as if SEMA3F re-expression acted on pathways targeting cell cycle. The next challenge was to assess the in vivo effect of endogenous SEMA3F re-expression. In two different in vivo experimental models, we observed a significant anti-tumoral effect of the re-expression of SEMA3F in STC-1 neuroendocrine tumor cells. This was shown by a statistically significant decrease in ‘primary’ tumor volumes after grafting in the spleen or in the subcutaneous tissue, as well as in the surface occupied by intra-hepatic liver nodules, obtained either through blood dissemination of intrasplenically grafted tumor cells or after metastatic dissemination from an intracaecal tumor graft. Our results also suggest that the antitumoral effect of SEMA3F re-expression is likely to be multifactorial and might involve a combination of anti-proliferative and anti-angiogenic effects, as shown by the decrease in Ki67 index and in microvascular surface within tumor tissue. In contrast, we could not detect any pro-apoptotic effect in tumor tissue, at least through the evaluation of caspase-3 activity.Our strategy was therefore to evaluate the impact of the re-expression of SEMA3F in STC-1 and GluTag cells in vitro and in vivo. In human tumors, mTOR pathway appeared strongly activated with tumor stage. A link between these effectors and proliferation has already been demonstrated both by molecular biology experiment but also by using specific inhibitors of mTOR pathway [We further attempted to uncover the intracellular signaling pathways affected by SEMA3F expression levels. Previous studies in other tumor types have shown that SEMA3F expression is associated with a reduction in activated MAPK signaling , 35. In  pathway . In our  pathway . Taken tin vitro, in association with growth inhibition of the xenografted tumors in vivo. Therefore, SEMA3F and its signaling pathway could represent a target for neuroendocrine tumor therapy.In summary, we have showed for the first time that the axon guidance factor SEMA3F was expressed in endocrine cells of the intestinal epithelium and was lost in neuroendocrine tumors arising in the distal small intestine, according to the stage. We also demonstrated that overexpression of SEMA3F reduced the proliferation of neuroendocrine cancer cells Tissue samples of GI-NETs were obtained from the collection of digestive neuroendocrine tumors housed in the tumor tissue bank “Tumorothèque des Hospices Civils de Lyon” , recognized by the French Ministry of Health (DC 2008-72 and AC 2008-73), supported by INCA and member of the BBMRI initiative; the biobank works in strict accordance with French ethical and regulatory issues.SEMA3F expression was studied in two tissue microarrays and in whole sections of ileal NETs. For expression screening, two tissue microarrays (TMA) were used. The first one contained cores from primary GI-NETs including 24 ileal NETs. The second one contained cores from primary NETs and all their available metastases, including 13 cases of ileal NETs along with their lymph node and liver metastases ; the 13 cases represented in the second TMA were also present in the first one. All 24 tumors were locally advanced and 22 were metastatic to the liver. For further detailed analysis of SEMA3F expression in tumor tissue, samples of 101 tumors from 38 patients with multifocal NETs of the distal small intestine , were ob2 selected in the areas of highest density of Ki67 labelled cells.In all cases, the neuroendocrine nature of the tumor was confirmed by the demonstration of chromogranin A and synaptophysin expression. All studied tumors were well differentiated. Their histological grade was determined according to current recommendations ; all tumFour-week old female Swiss nu/nu mice  were housed and bred in the pathogen free animal facility “AniCan” (Lyon). Experiments were performed in accordance with animal care guidelines of the European Union and French laws and were approved by the local Animal Ethic Evaluation Committee (CECCAPP).6 STC-1 cells were injected into the spleen, from where they disseminated into the liver. The first group (11 mice STC-AP and 11 STC-S3F2) was sacrificed at day 7 while the second (15 STC-AP and 16 STC- S3F2) was sacrificed at day 28. Both groups were weighted twice a week, and after sacrifice, livers were weighted and prepared for histological analysis.The xenografting procedure was as previously described . Briefly6 STC-AP or STC-S3F2 cells into dorsolateral flanks of anesthetized nude mice. Tumor volume was measured at 15, 22, 29 and 32 days after the injection. Then, subcutaneous tumor fragments were orthotopically xenografted into the caecal wall. A total of 7 mice were intracaecally grafted . Mice were sacrificed 8 weeks after grafting. Intracaecal tumors and livers were recovered.The xenografting procedure was performed in 2 steps as previously described . First, Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. For histological examination, 4 μm-thick sections were stained with hematoxylin-phloxin-saffron and observed with a light microscope. The amount of intrahepatic tumor tissue was evaluated by morphometry using Histolab software (Alphelys). The microvascular surface within intrahepatic nodules was assessed by measuring the surface occupied by all structures expressing the endothelial cell marker CD31. The Ki67 index was determined as the percentage of positive cells within intrahepatic nodules.The STC-1 and the GluTag cell lines were cultured in DMEM supplemented with 5% (STC-1) or 10% (GluTag) fetal calf serum (FCS), 2 mM glutamine and antibiotics  , 21. TheFor immunoblot, antibodies to SEMA3F (ab39956) and NRP-1 (ab81321) were purchased from Abcam . Antibodies to NRP-2 (#3366), PLEXIN-A1 (#3813), Myc-Tag (#2276), phospho-ERK (#4370), ERK (#9102), phospho-p70S6K (#9234), p70S6K (#9202) and cleaved caspase-3 (#9664) were from Cell Signaling Technology  and the antibody to α-Tubulin (T6074) was from Sigma-Aldrich .For immunohistochemistry, the antibody for SEMA3F (HP035008) was purchased from Atlas Antibodies. Anti-phospho-p70S6K (sc-7984) was from Santa Cruz Biotechnology . Anti-chromogranin A (M0869), anti-human Ki67  and anti-mouse Ki67  were from Dako . Anti-CD31 antibody was from AnaSpec (#53332) . For immunofluorescence, the antibody to phospho-Histone H3 (pH-H3) was from Cell Signaling Technology (#9706).et al. [DNA from tumor tissues was extracted from 18 formalin-fixed paraffin-embedded ileal NETs according to manufacturer's instructions . DNA modification was performed according to manufacturer's instructions  then MSP was performed as described by Herman et al. .2O2 for 10 minutes. An enhanced streptavidin-biotin staining procedure was followed: sections were incubated with the appropriate biotinylated antibody for 30 minutes at room temperature; revelation was carried out using the EnVision™ detection kit (Dako) according to manufacturer's instructions; sections were then counterstained with Meyer's hematoxylin.Both human and mouse tissue sections were subjected to antigen retrieval, then treated with 3% HTissue sections and cell lines grown on glass coverslips were incubated with the primary antibodies respectively for 30 minutes at room temperature or overnight at 4°C. Both cells and sections were then incubated with secondary Alexa Fluor 488 or −568-conjugated antibodies  for 1 hour and counterstained with DAPI. Finally, pictures were taken using a Leica SP5X confocal laser scanning microscope or a Zeiss inverted microscope Axio Observer D1.Cells were seeded and maintained 72 hours under normal culture conditions, then lysed in RIPA buffer plus protease and phosphatase inhibitors (Santa Cruz Biotechnologies). The membranes were hybridized with primary antibodies overnight at 4°C. Immunodetection was performed using electrochemiluminescence  and the ChemiDoc XR5 machine .SEMA3F-MYC and the corresponding empty vector pSectag-AP were kindly provided by Dr. David Ginty  and Dr Joëlle Roche . Briefly, the full-length SEMA3F cDNA fragment lacking stop codons was ligated into the pSectag expression vector (Life Technologies). The full length SEMA3F was placed between an APtag sequence at N-terminal and a MYC tag sequence at the C-terminal ending [SEMA3F-MYC and pSectag-AP plasmids were transfected into STC-1 and GluTag cells by using the SuperFect Transfection Reagent and selected for stable expression with 300 μg/mL zeocin (Life Technologies).The SEMA3F expression plasmid AP-l ending . Both APSEMA3F-MYC vector were grown in 25 cm3 tissue flasks with medium devoided of FCS and antibiotics. Then, the culture supernatant was recovered and concentrated by size exclusion filtration by using a 30 kDa Amicon Ultra centrifugal filter unit (Millipore). RBA was performed following the procedure described by Brennan et al. [We used receptor-binding assay (RBA) staining method to demonstrate SEMA3F ectodomain binding within our cell lines. Selected stable clones containing the pSectag-AP empty vector or the AP-n et al. .Cell survival and proliferation were assessed by MTT method. The proliferative capacities of transfected cells were evaluated by phospho-Histone H3 (pH-H3) immunostaining. Cell cycle analysis was performed using propidium iodide-based Cycletest™ Plus-DNA Reagent Kit  and Canto II cytometer.t-test or Chi-squared test. A P value inferior to 0.05 was considered as significant.All data were mean values of at least three individual experiments and were expressed as mean ± SEM. Differences between means were compared by unpaired two-tailed student's