Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

A Wnt/ß-catenin signaling inhibitor, IMU1003, suppresses the emergence of osimertinib-resistant colonies from gefitinib-resistant non-small cell lung cancer cells.

Drug resistance has become a challenge in effective longterm molecular targeted therapy. Longterm non-small cell lung cancer (NSCLC) treatments with the first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) shorten the effective duration of the third-generation EGFR-TKI, osimertinib, via genetic or epigenetic mechanisms in addition to the gatekeeper mutation T790M. This study reproduced this persistence in vitro using gefitinib-resistant NSCLC PC-9 cells (GR cells) and revealed that pharmacological nuclear localization inhibition of ß-catenin suppressed the osimertinib resistance. Osimertinib effectively reduced GR cell survival but left significantly more resistant colonies than parental PC-9 cells. The nuclear fraction of ß-catenin was enriched in GR cells during acquisition of osimertinib resistance. A chemical nuclear localization inhibitor of ß-catenin, IMU1003, dramatically decreased the emergence of osimertinib-resistant colonies. Forced nuclear localization of ß-catenin reduced IMU1003 efficacy. Thus, suppression of the nuclear ß-catenin function may overcome the transgenerational EGFR-TKI-resistance.

Domestication of chemicals attacking metazoan embryogenesis: identification of safe natural products modifying developmental signaling pathways in human.

Soil microorganisms are rich sources of bioactive natural products. Interspecies interactions are the cues of their production and refine biological activities. These interactions in natural environments include the interplay between microorganisms and Metazoans (animals), such as nematodes, insects, and ticks. Chemical intercellular communication modulators could exert ideal Metazoan-selective toxicity for defending microorganisms. Developmental signaling pathways, such as the Notch, TGF-beta, and Wnt pathways, are intercellular communication networks that contribute to the reproducible formation of complex higher-order Metazoan body structures. Natural modifiers of the developmental signaling pathway are attractive therapeutic seeds for carcinoma and sarcoma treatment. However, these fundamental signaling pathways also play indispensable physiological roles and their perturbation could lead to toxicity, such as defects in stem cell physiology and tissue regeneration processes. In this review, we introduce a screening system that selects developmental signaling inhibitors with wide therapeutic windows using zebrafish embryonic phenotypes and provide examples of microorganism-derived Wnt pathway inhibitors. Moreover, we discuss safety prospects of the developmental signaling inhibitors.

Switching from Intravenous to Oral Tacrolimus Reduces its Blood Concentration in Paediatric Cancer Patients.

BACKGROUND/AIM: Tacrolimus is an essential immunosuppressant for successful allogeneic haematopoietic stem cell transplantation (Allo-HSCT). This study aimed to examine the change in the blood concentration of tacrolimus during switching from intravenous to oral administration in allo-HSCT for paediatric cancer to predict the optimal dosage. PATIENTS AND METHODS: We retrospectively examined the medical records of 63 patients who received allo-HSCT and were administered tacrolimus. To compare bioavailability among different dose ranges, the blood concentration was divided by the dose (C/D). RESULTS: Thirty-nine patients (age range=children 1-15 years, adults 17-67 years) were switched to oral administration of tacrolimus. The C/D after switching was significantly lower in children than in adults (p=0.039). There was a strong positive correlation between age and C/D in children, whereas no correlation was observed in adults. CONCLUSION: In paediatric cancer patients, switching tacrolimus administration route may result in reduced blood concentrations. This tendency is more prominent in younger children.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

Synthesis and evaluation of azalamellarin N and its A-ring-modified analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant.

Azalamellarin N, a synthetic lactam congener of the marine natural product lamellarin N, and its A-ring-modified analogues were synthesized and evaluated as potent and non-covalent inhibitors of the drug-resistant epidermal growth factor receptor T790M/L858R mutant. An in vitro tyrosine kinase assay indicated that the inhibitory activities of the synthetic azalamellarin analogues were higher than those of the corresponding lamellarins. The azalamellarin analogue bearing two 3-(dimethylamino)propoxy groups at C20- and C21-positions exhibited the highest activity and selectivity against the mutant kinase [IC50 (T790M/L858R) = 1.7 nM; IC50 (WT) = 4.6 nM]. The inhibitory activity was attributed to the hydrogen bonding interaction between the lactam NH group of the B-ring and carbonyl group of a methionine residue.

IMU1003, an atrarate derivative, inhibits Wnt/ß-catenin signaling.

Aberrant activation of the canonical Wnt/ß-catenin signaling pathway triggers tumorigenesis in various tissues. This study identified an atrarate compound, IMU14, derived from filamentous fungi as an inhibitor of Wnt/ß-catenin signaling in phenotypic chemical inhibitor screening of the zebrafish eyeless phenotype. Its derivatization resulted in synthesis of IMU1003 with enhanced Wnt inhibitory activity. IMU1003 inhibited ß-catenin/TCF-dependent transcriptional activation and decreased nuclear ß-catenin level. In addition, IMU1003 selectively decreased viability and target gene products of the Wnt/ß-catenin signaling pathway in human non-colorectal cancer cell lines harboring intact APC and ß-catenin. Therefore, atrarate derivatives inhibit Wnt/ß-catenin signaling and show anticancer potential, and we developed a new class of chemical backbones for Wnt/ß-catenin signaling inhibitors.

Bucillamine Prevents Afatinib-Mediated Inhibition of Epidermal Growth Factor Receptor Signaling.

Molecular targeting therapies often cause characteristic adverse effects, such as skin rash during anti-epidermal growth factor receptor (EGFR) therapies, making treatment continuation difficult. In contrast, skin symptoms induced by EGFR inhibition are strongly correlated with the overall survival of the therapies. Therefore, controlling adverse effects not only facilitates treatment continuation but also increases clinical benefits. In this study, we proposed a novel strategy for reducing EGFR-tyrosine kinase inhibitor (TKI)-induced adverse effects in nontumorous organs by repositioning approved medicines using a zebrafish model. We developed a model system for evaluating chemical quenchers of afatinib, a clinically available irreversible EGFR-TKI, by scoring the inhibition of afatinib-induced hyperformation of lateral line neuromasts in zebrafish larvae. Bucillamine, an antirheumatic drug, was identified as an afatinib quencher in the zebrafish system and inhibited TKI activity in vitro. In addition, bucillamine restored EGFR autophosphorylation and downstream signaling in afatinib-treated A431 cells. Thus, topical bucillamine is a potential reliever of irreversible EGFR-TKI-induced skin rash. The zebrafish model can be applied to a screening for quenchers of other anti-EGFR-targeting therapies, including reversible TKIs and biologics.

Clotrimazole inhibits the Wnt/ß-catenin pathway by activating two eIF2α kinases: The heme-regulated translational inhibitor and the double-stranded RNA-induced protein kinase.

The Wnt/ß-catenin signaling pathway controls cell proliferation and differentiation, and therefore, when this pathway is excessively activated, it causes tumorigenesis. Our chemical suppressor screening in zebrafish embryos identified antifungal azoles including clotrimazole, miconazole, and itraconazole, as Wnt/ß-catenin signaling inhibitors. Here we show the mechanism underlying the Wnt/ß-catenin pathway inhibition by antifungal azoles. Clotrimazole reduced ß-catenin revels in a proteasome-independent fashion. By gene knockdown of two translational regulators, heme-regulated translational inhibitor and double-stranded RNA-induced protein kinase, we show that they mediate the clotrimazole-induced inhibition of the Wnt/ß-catenin pathway. Thus, clotrimazole inhibits the Wnt/ß-catenin pathway by decreasing ß-catenin protein levels through translational regulation. Antifungal azoles represent genuine candidate compounds for anticancer drugs or chemopreventive agents that reduce adenomatous polyps.

Sensitisation of Cancer Cells to MLN8237, an Aurora-A Inhibitor, by YAP/TAZ Inactivation.

BACKGROUND: Transcriptional co-activators YES-associated protein (YAP) and transcriptional coactivator with PDZ-motif (TAZ) stimulate the expression of cell cycle-related genes to permit for tumour cell growth. MLN8237 is a potent aurora-A kinase inhibitor; however, patients responding to MLN8237 are limited. Therefore, rational combination therapy could enhance their response. MATERIALS AND METHODS: YAP and TAZ were depleted using siRNA and then treated with MLN8237 in YAP/TAZ-dependent OVCAR-8 and MDA-MB-231 cell lines. MLN8237 was combined with fluvastatin, an agent constraining nuclear localisation of YAP/TAZ for potential combination therapy in vitro. RESULTS: Depletion of either YAP or TAZ sensitised these cell lines to MLN8237, resulting in apoptosis and reduction in aurora-A. MLN8237 reduced YAP/TAZ expression. A combination of MLN8237 with fluvastatin effectively reduced the cell viability of OVCAR-8 and MDA-MB-231 cell lines. CONCLUSION: A combination of MLN8237 and small-molecule agents inactivating YAP/TAZ, such as statins, could be a novel therapeutic strategy for YAP/TAZ-dependent cancer.

Augmentation of the therapeutic efficacy of WEE1 kinase inhibitor AZD1775 by inhibiting the YAP-E2F1-DNA damage response pathway axis.

The main reasons for failure of cancer chemotherapy are intrinsic and acquired drug resistance. The Hippo pathway effector Yes-associated protein (YAP) is associated with resistance to both cytotoxic and molecular targeted drugs. Several lines of evidence indicate that YAP activates transcriptional programmes to promote cell cycle progression and DNA damage responses. Therefore, we hypothesised that YAP is involved in the sensitivity of cancer cells to small-molecule agents targeting cell cycle-related proteins. Here, we report that the inactivation of YAP sensitises the OVCAR-8 ovarian cancer cell line to AZD1775, a small-molecule WEE1 kinase inhibitor. The accumulation of DNA damage and mitotic failures induced by AZD1775-based therapy were further enhanced by YAP depletion. YAP depletion reduced the expression of the Fanconi anaemia (FA) pathway components required for DNA repair and their transcriptional regulator E2F1. These results suggest that YAP activates the DNA damage response pathway, exemplified by the FA pathway and E2F1. Furthermore, we aimed to apply this finding to combination chemotherapy against ovarian cancers. The regimen containing dasatinib, which inhibits the nuclear localisation of YAP, improved the response to AZD1775-based therapy in the OVCAR-8 ovarian cancer cell line. We propose that dasatinib acts as a chemosensitiser for a subset of molecular targeted drugs, including AZD1775, by targeting YAP.

Design, synthesis, and evaluation of A-ring-modified lamellarin N analogues as noncovalent inhibitors of the EGFR T790M/L858R mutant.

A series of A-ring-modified lamellarin N analogues were designed, synthesized, and evaluated as potential noncovalent inhibitors of the EGFR T790M/L858R mutant, a causal factor in the drug-resistant non-small cell lung cancer. Several water-soluble ammonium- or guanidinium-tethered analogues exhibited good kinase inhibitory activities. The most promising analogue, 14f, displayed an excellent inhibitory profile against the T790M/L858R mutant [IC50 (WT) = 31.8 nM; IC50 (T790M/L858R) = 8.9 nM]. The effects of A-ring-substituents on activity were rationalized by docking studies.

Dynamic Phenotypic Transition of Breast Cancer Cells In Vitro Revealed by Self-floating Cell Culture.

BACKGROUND: Breast tumor heterogeneity leads to phenotypic diversity, such as tumor-initiating and metastatic properties and drug sensitivity. MATERIALS AND METHODS: We found that a self-floating cell (SFC) culture enriches a drug-resistant subpopulation in a HER2-positive breast cancer cell line. SFCs were analyzed for cancer stem cell markers, gene expression profiles, and sensitivity for anticancer drugs. RESULTS: SFCs expressed cancer stem cell markers, such as aldehyde dehydrogenase (ALDH) activity and elevated HER2 autophosphorylation. Gene expression profiles of SFCs showed a dramatic difference compared to those of parental or forced floating cells. SFCs also expressed CD133, a marker of drug resistance, and resisted cytotoxic drugs by drug efflux transporters. In contrast, HER2 kinase inhibitors efficiently reduced SFC viability. CONCLUSION: SFCs enrich drug-resistant subpopulations even in vitro and might reflect the highly plastic nature of breast cancer cells even in vitro.

Screening for Chemical Suppressors of the Wnt/ß-catenin Signaling Pathway.

Aberrant activation of Wnt/ß-catenin canonical signaling is observed in multiple malignant tumors, and is recognized as an attractive therapeutic target for molecular targeted drugs. This signaling pathway is also involved in maintaining pluripotency in adult stem cells. Therefore, lowering potential stem cell toxicity is a key factor for the development of a Wnt/ß-catenin signaling inhibitor. Here, we show Wnt/ß-catenin pathway inhibitors with low toxicity, identified through phenotype-based screening using zebrafish embryos. Artificial activation of the Wnt/ß-catenin pathway in fertilized eggs, which are often considered the "ultimate stem cells", results in an "eyeless" phenotype in zebrafish embryos. Screening for compounds that rescue this "eyeless" phenotype and have no effects on normal embryogenesis could help us identify Wnt/ß-catenin pathway inhibitors with minimal stem cell toxicities, at least at a concentration that suppresses aberrant signaling. Chemical suppressors of the "eyeless" phenotype include novel and known compounds with different modes of action. Some of these compounds diminish the activation of crosstalk between other signaling pathways and the Wnt/ß-catenin pathway. These inhibitors reduced tumor growth in ApcMin/+ mice and did not show apparent toxicities. Thus, our screening for chemical suppressors of the "eyeless" phenotype allowed us to successfully identify inhibitors for the Wnt/ß-catenin pathway with low toxicity.

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M. CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers.

YAP and TAZ oncoproteins confer malignancy and drug resistance to various cancer types. We screened for small molecules that inhibit the nuclear localization of YAP/TAZ. Dasatinib, statins and pazopanib inhibited the nuclear localization and target gene expression of YAP and TAZ. All three drugs induced phosphorylation of YAP and TAZ, and pazopanib induced proteasomal degradation of YAP/TAZ. The sensitivities to these drugs are correlated with dependence on YAP/TAZ in breast cancer cell lines. Combinations of these compounds with each other or with other anti-cancer drugs efficiently reduced cell proliferation of YAP/TAZ-dependent breast cancer cells. These results suggest that these drugs can be therapeutics and chemosensitizers for YAP/TAZ-dependent breast cancers.

A zebrafish chemical suppressor screening identifies small molecule inhibitors of the Wnt/ß-catenin pathway.

Genetic screening for suppressor mutants has been successfully used to identify important signaling regulators. Using an analogy to genetic suppressor screening, we developed a chemical suppressor screening method to identify inhibitors of the Wnt/ß-catenin signaling pathway. We used zebrafish embryos in which chemically induced ß-catenin accumulation led to an "eyeless" phenotype and conducted a pilot screening for compounds that restored eye development. This approach allowed us to identify geranylgeranyltransferase inhibitor 286 (GGTI-286), a geranylgeranyltransferase (GGTase) inhibitor. Our follow-up studies showed that GGTI-286 reduces nuclear localization of ß-catenin and transcription dependent on ß-catenin/T cell factor in mammalian cells. In addition to pharmacological inhibition, GGTase gene knockdown also attenuates the nuclear function of ß-catenin. Overall, we validate our chemical suppressor screening as a method for identifying Wnt/ß-catenin pathway inhibitors and implicate GGTase as a potential therapeutic target for Wnt-activated cancers.

Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents.

Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs.

Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity.

Slit-Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4-paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2-Robo4-paxillin-GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.

Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability.

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.