{"gene":"ROBO1","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2001,"finding":"Targeted deletion of exon 5 (eliminating the first Ig domain) of Dutt1/Robo1 in mice causes neonatal respiratory failure due to delayed lung maturation (reduced air spaces, increased mesenchyme) and bronchial epithelial hyperplasia in survivors, establishing a direct functional role for Robo1 in lung development.","method":"Targeted gene knockout in mice (homozygous deletion of exon 5); histopathology and phenotypic analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout mouse model with specific developmental phenotype, replicated across homozygous and survivor cohorts in a single rigorous study","pmids":["11734623"],"is_preprint":false},{"year":2004,"finding":"Dutt1/Robo1 heterozygous mice spontaneously develop lymphomas and lung adenocarcinomas; tumors retain the structurally normal allele but show promoter methylation and reduced Robo1 protein, consistent with Knudson two-hit tumor suppressor mechanism.","method":"Heterozygous knockout mouse model; LOH analysis; bisulfite sequencing of promoter; Western blot and immunohistochemistry for protein expression","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (LOH, methylation, protein expression) in a genetic mouse model within a single study","pmids":["15374951"],"is_preprint":false},{"year":2002,"finding":"The DUTT1/ROBO1 promoter CpG island is hypermethylated in breast, lung, and renal cell carcinoma tumors; methylation silences expression, which is reactivated by 5-aza-2'-deoxycytidine treatment, identifying promoter methylation as an epigenetic inactivation mechanism.","method":"Bisulfite sequencing; methylation-specific PCR; RT-PCR expression analysis; 5-aza-2'-deoxycytidine demethylation assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal epigenetic and expression methods, replicated across multiple cancer types and cell lines","pmids":["12082532"],"is_preprint":false},{"year":2006,"finding":"Robo1 knockout mice display major forebrain axon pathfinding defects (corpus callosum and hippocampal commissure dysgenesis, premature corticothalamic/thalamocortical targeting) and up to twice as many cortical interneurons migrate into the cortex, establishing Robo1 as a regulator of both major axon tract formation and interneuron migration.","method":"Robo1 knockout mice (targeted deletion of exon 5); MRI diffusion tensor imaging; immunohistochemistry; cell counting","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with multiple distinct phenotypic readouts (axon guidance and cell migration) replicated across developmental time points","pmids":["16690755"],"is_preprint":false},{"year":2007,"finding":"RNAi-mediated knockdown of Robo1 in glioma cell lines abolishes the repulsive/migratory-inhibitory effect of recombinant Slit2, demonstrating that Robo1 is the major functional receptor for Slit2-mediated chemorepulsion in glioma cells.","method":"siRNA knockdown of Robo1; modified Boyden chamber migration assay with recombinant Slit2","journal":"Journal of neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional rescue/loss-of-function assay with defined phenotypic readout, single lab, single method","pmids":["17968499"],"is_preprint":false},{"year":2007,"finding":"The C. elegans kinesin-related protein VAB-8L promotes posterior cell/growth-cone migration by increasing surface levels of the Robo1 ortholog SAX-3; the Rac GEF UNC-73 (Trio) physically interacts with both VAB-8L and the intracellular domain of SAX-3, supporting a model in which receptor surface localization is regulated by this complex.","method":"C. elegans genetics (epistasis); co-immunoprecipitation; receptor abundance measurement; gain/loss-of-function assays","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic epistasis combined with physical interaction (co-IP) and receptor localization data in a single rigorous study","pmids":["17237778"],"is_preprint":false},{"year":2008,"finding":"Robo1 forms a heterodimeric complex with Robo4; Robo1 is required for Robo4-mediated filopodia induction in endothelial cells; the Robo4 intracellular domain binds WASP, N-WASP, and WIP (actin nucleation-promoting factors) as identified by yeast two-hybrid and GST pulldown, linking Robo1/Robo4 signaling to actin-based cell motility.","method":"siRNA knockdown; Robo4-GFP overexpression; yeast two-hybrid; GST pulldown; endothelial tube formation and migration assays; co-immunoprecipitation","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, GST pulldown, siRNA functional assay, co-IP) in a single study establishing complex formation and downstream actin effectors","pmids":["18948384"],"is_preprint":false},{"year":2010,"finding":"Robo1 and Robo2 have distinct and collaborative roles in post-crossing commissural axon guidance: Robo2 is required for axons to project away from the floor plate into the lateral funiculus, while Robo1 prevents axonal stalling after midline crossing; genetic epistasis with Robo3 mutants further defines their collaborative role in preventing midline re-entry.","method":"Genetic combination mutants (Robo1, Robo2, Robo3 single and double/triple knockouts); axon trajectory analysis in mouse spinal cord","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis with multiple allele combinations defining distinct roles of each paralog","pmids":["20631173"],"is_preprint":false},{"year":2010,"finding":"Human ROBO1 undergoes sequential proteolytic cleavage by metalloproteinases (producing ROBO1-CTF1 at 129 kDa) followed by gamma-secretase cleavage (producing ROBO1-CTF2 at 118 kDa); the resulting intracellular fragment accumulates in the nucleus in cancer cells, and nuclear accumulation is abolished by either metalloproteinase inhibitor TAPI-1 or gamma-secretase inhibitor L-685,458.","method":"Western blot of cleavage fragments; pharmacological inhibition with TAPI-1 and L-685,458; nuclear fractionation; determination of precise cleavage site","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical identification of cleavage products with pharmacological validation, single lab","pmids":["20471383"],"is_preprint":false},{"year":2011,"finding":"SLIT/ROBO1 signaling in the mammary basal cell layer restricts basal cell proliferation by inhibiting canonical WNT signaling, shifting β-catenin from the nuclear pool to cytoplasmic/membrane pools; loss of SLIT/ROBO1 signaling specifically in basal cells results in excess basal cells and precocious branching morphogenesis. TGF-β1 acts upstream of Robo1, inducing its expression in the basal layer.","method":"Conditional loss-of-function mouse models; mammary gland whole-mount analysis; immunofluorescence for β-catenin localization; Western blot; TGF-β1 treatment","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional knockout with mechanistic pathway dissection (WNT/β-catenin), multiple orthogonal methods","pmids":["21664580"],"is_preprint":false},{"year":2013,"finding":"Slit2-Robo1 signaling inhibits Cdc42 activity in glioma cells, and this is the mechanism by which the pathway suppresses glioma cell invasion; Cdc42-GTP inactivation mediates the anti-invasive effect.","method":"Small GTPase pull-down assay (Cdc42 activity); siRNA knockdown; migration/invasion assay","journal":"Neuroscience bulletin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single GTPase activity assay with no independent replication; abstract-level detail only","pmids":["21113198"],"is_preprint":false},{"year":2013,"finding":"Heparin binds to Robo1 with an affinity of ~650 nM; N-sulfo and 6-O-sulfo groups on heparin are essential for the Robo1-heparin interaction; full-length heparin chains and higher sulfation levels increase binding affinity.","method":"Surface plasmon resonance (SPR); chemical modification of heparin; SPR solution competition studies","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro binding assay with systematic chemical modification, single lab","pmids":["23994753"],"is_preprint":false},{"year":2014,"finding":"FLRT3 is a novel co-receptor for Robo1; in thalamic axons, Slit1 acting through both Robo1 and FLRT3 induces Netrin-1 attraction by upregulating surface DCC via activation of protein kinase A (PKA); thalamic axons lacking FLRT3 are insensitive to Netrin-1, and loss of FLRT3 causes axon guidance defects in vivo.","method":"Co-receptor identification; in vitro axon guidance assays; PKA activation assays; surface DCC measurement; in vivo guidance phenotype analysis in knockout mice","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods establishing a novel co-receptor interaction with defined downstream signaling (PKA/DCC axis)","pmids":["24560577"],"is_preprint":false},{"year":2014,"finding":"ROBO1 negatively regulates cell motility and invasiveness of prostate cancer cells through interaction with DOCK1 (via its SH3-SH2 domain), controlling Rac1 activation; loss of ROBO1 leads to Rac1 activation, which destabilizes E-cadherin/β-catenin cytoskeleton and induces cell migration.","method":"ROBO1-expressing and ROBO1-C2C3 mutant constructs; co-immunoprecipitation; confocal microscopy; luciferase reporter; migration/invasion assays","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain-mapping mutants and functional assays, single lab","pmids":["24752651"],"is_preprint":false},{"year":2014,"finding":"Robo1 knockout in the developing neocortex causes a significant increase in progenitor cells and both early- and late-born pyramidal neuron populations due to prolonged proliferative activity of progenitors; confirmed by in utero electroporation of Robo1 shRNA and in dissociated cortical cell cultures.","method":"Robo1 knockout mice; in utero electroporation with shRNA; dissociated cortical cell cultures; BrdU/Ki67 proliferation analysis; layer-specific marker immunostaining","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout replicated by independent shRNA electroporation and in vitro assays within a single study","pmids":["24741061"],"is_preprint":false},{"year":2015,"finding":"Slit2 acting through Robo1 and Robo2 promotes endothelial cell migration and retinal angiogenesis; Robo1 and Robo2 are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation in retinal endothelial cells.","method":"Conditional knockout mice (endothelial-specific deletion of Slit2, Robo1, Robo2); mouse postnatal retinal angiogenesis assay; Rac1 activation assay; lamellipodia measurement; ocular neovascular disease model","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockouts with in vivo and in vitro mechanistic readouts including Rac1 activation and lamellipodia formation","pmids":["25894826"],"is_preprint":false},{"year":2015,"finding":"Two distinct heparin-binding sites on Robo1 were identified: one at the previously known site and a second at the disordered N-terminus; mutagenesis of the N-terminal site decreased heparin binding affinity; heparin binding also induces a minor conformational change in the Ig2 domain.","method":"Electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting; surface plasmon resonance; heparin affinity chromatography; site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — novel structural footprinting method combined with mutagenesis and SPR quantification in a single rigorous study","pmids":["25752613"],"is_preprint":false},{"year":2015,"finding":"SLIT2/ROBO1 signaling regulates mammary stem cell self-renewal by governing the choice between asymmetric and symmetric cell divisions through the transcription factor SNAI1 and spindle orientation protein mINSC; loss of SLIT2/ROBO1 increases nuclear SNAI1, which upregulates mInsc, increasing symmetric divisions and expanding the stem cell pool.","method":"Conditional knockout mouse model; single-cell lineage tracing; mInsc/SNAI1 expression analysis; SLIT2 treatment rescue experiments; mammary gland outgrowth assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional genetic model with mechanistic pathway dissection (SNAI1/mInsc axis) and rescue experiments","pmids":["26440891"],"is_preprint":false},{"year":2015,"finding":"Activation of Slit2-Robo1 signaling promotes liver fibrosis by activating hepatic stellate cells (HSCs); mechanistically, Robo1 signaling phosphorylates Smad2, Smad3, PI3K, and AKT independently of TGF-β1, and promotes α-SMA expression; Robo1 neutralizing antibody R5 and Robo1 siRNA block these effects in vitro and attenuate CCl4-induced fibrosis in vivo.","method":"Slit2 transgenic and Robo1/2 heterozygous knockout mice; CCl4-induced fibrosis model; Robo1 siRNA and neutralizing antibody (R5); Western blot for Smad2/3, PI3K, AKT, α-SMA phosphorylation","journal":"Journal of hepatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models (transgenic, knockout, siRNA, antibody) with defined signaling pathway readouts in a single study","pmids":["26264936"],"is_preprint":false},{"year":2015,"finding":"Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway; mechanistically, Robo1 activation by Slit2 activates Src, which downregulates E-cadherin, thereby releasing β-catenin to activate Wnt target genes.","method":"Slit2 transgenic and ApcMin/+ mouse models; DMH/DSS colorectal carcinoma model; Western blot; immunohistochemistry; pharmacological Src inhibition","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vivo mouse models with defined signaling pathway (Src/E-cadherin/β-catenin), single lab","pmids":["25605242"],"is_preprint":false},{"year":2015,"finding":"srGAP1 is a Robo1-interacting protein that mediates the anti-migratory function of Slit2-Robo1 in colorectal cancer cells by inhibiting Cdc42 activity; co-immunoprecipitation confirmed the Robo1-srGAP1 interaction; immunofluorescence showed similar dynamic subcellular distribution upon Slit2 treatment.","method":"Co-immunoprecipitation; immunofluorescence; Cdc42 GTPase pull-down assay; wound healing migration assay","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating physical interaction with functional GTPase activity assay, single lab","pmids":["27923383"],"is_preprint":false},{"year":2015,"finding":"Recombinant Slit2 reduces peripheral immune cell infiltration after surgical brain injury through Robo1-srGAP1-mediated inhibition of Cdc42 activity; decoy Robo1 co-administration and srGAP1 siRNA reversed the protective effects of Slit2, establishing the Robo1-srGAP1-Cdc42 signaling axis in immune cell migration.","method":"Rat surgical brain injury model; recombinant Slit2 and decoy Robo1; srGAP1 siRNA; Cdc42 activity assay; brain edema measurement; immunohistochemistry","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo model with decoy receptor and siRNA controls, Cdc42 activity assay; single lab","pmids":["26550694"],"is_preprint":false},{"year":2015,"finding":"Slit2 promotes angiogenesis via the Robo1-VEGFR2-ERK1/2 pathway; Slit2 overexpression upregulates Robo1, VEGFR2, and phosphorylated ERK1/2 in both transgenic mice and HUVECs, and promotes endothelial cell proliferation, migration, and tube formation.","method":"Slit2 transgenic mice; laser-induced CNV model; HUVEC transfection; Western blot for VEGFR2 and p-ERK1/2; proliferation, migration and tube formation assays","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic mouse model combined with in vitro pathway analysis, single lab","pmids":["26244297"],"is_preprint":false},{"year":2016,"finding":"Heparan sulfate octasaccharide ligand requirements for Robo1 were defined: Robo1 preferentially binds structures with 6-O-sulfate esters (critical), whereas 2-O-sulfate esters substantially reduce binding; a high-affinity HS octasaccharide ligand was synthesized and confirmed by SPR to bind Robo1 and reverse Slit2-Robo1-mediated inhibition of endothelial cell migration.","method":"HS affinity enrichment; HILIC-MS/MS structural determination; chemical synthesis of HS oligosaccharides; surface plasmon resonance (SPR) binding studies; endothelial cell migration assay","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Strong — integrated chemical synthesis with quantitative SPR binding and functional cell migration assay, rigorous structure-activity relationship study","pmids":["27611601"],"is_preprint":false},{"year":2016,"finding":"NMR characterization of Robo1-Ig1-2 interaction with a synthetic heparan sulfate tetramer identified K81 as part of the binding site (Kd ~255 µM); saturation transfer difference and trNOE defined the binding epitope and bound conformation of HS; paramagnetic perturbation localized the binding site; a structural model of the complex was generated.","method":"Single amino acid type NMR labeling; STD NMR; trNOE; paramagnetic perturbation; complex modeling","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple NMR methods characterizing binding site, bound conformation, and dissociation constant; single lab","pmids":["27653286"],"is_preprint":false},{"year":2016,"finding":"A stiff ECM increases Robo1 levels by downregulating miR-203; SLIT2/ROBO1 signaling stimulates Rac activity and focal adhesion kinase (FAK) to enhance cell tension, maintain cell shape, and facilitate matrix adhesion; Robo1 ablation in the mammary gland compromises actin stress fiber assembly and inhibits cell contractility.","method":"miR-203 and Robo1 manipulation; Rac activation assay; FAK activity assay; actin staining; mammary gland conditional knockout; tunable hydrogel substrates","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro with multiple signaling readouts (Rac, FAK, actin); single lab","pmids":["26975850"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of Robo1 Ig1-4 and negative-stain EM reconstruction of the full ectodomain show that Robo1 forms compact dimers mediated by central Ig domains, which further interact in a 'back-to-back' fashion to generate tetrameric assemblies; no change in oligomerization is observed upon Slit2-N binding, suggesting Slit2-N induces a conformational change rather than oligomerization change.","method":"X-ray crystallography (Robo1 Ig1-4 and Ig5); negative stain electron microscopy; fluorescent imaging with dimeric Slit2-N","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with EM reconstruction and Slit2 binding experiment; single lab but Tier 1 methods","pmids":["29307485"],"is_preprint":false},{"year":2018,"finding":"SLIT2/ROBO1 axis promotes the Warburg effect (aerobic glycolysis) in osteosarcoma cells via activation of the SRC/ERK/c-MYC/PFKFB2 signaling pathway; knockdown or overexpression experiments in vitro and in vivo confirmed this metabolic-oncogenic role.","method":"siRNA knockdown and overexpression; xenograft mouse model; Western blot for SRC, ERK, c-MYC, PFKFB2; metabolic assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo models with defined signaling pathway; single lab","pmids":["29523788"],"is_preprint":false},{"year":2018,"finding":"USP33 (ubiquitin-specific protease 33) deubiquitinates and stabilizes Robo1; reduced USP33 in gastric cancer leads to increased Robo1 ubiquitination and degradation, diminishing the inhibitory effect of Slit2-Robo1 signaling on cell migration and EMT.","method":"Co-immunoprecipitation; ubiquitination protein analysis; siRNA knockdown; in vitro and in vivo migration/EMT assays","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating physical interaction and ubiquitination assay establishing USP33 as a deubiquitinase for Robo1; single lab","pmids":["30896071"],"is_preprint":false},{"year":2018,"finding":"miR-92 suppresses Robo1 translation (without mRNA degradation) in commissural neurons by binding the Robo1 3'UTR; disrupting miR-92/Robo1 interaction induces premature Slit2 repulsion and causes commissural axon projection defects in vivo; this mechanism regulates the temporal control of Slit sensitivity during midline crossing.","method":"miR-92 overexpression and inhibition; luciferase 3'UTR reporter assay; in vitro Slit2 repulsion assay; in vivo commissural axon labeling in chick embryo","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — 3'UTR reporter validation combined with in vitro repulsion assay and in vivo axon guidance phenotype in a single study","pmids":["30184503"],"is_preprint":false},{"year":2019,"finding":"Alternative splicing at a conserved microexon in mammalian ROBO1 and ROBO2, regulated by NOVA splicing factors, generates isoforms with distinct guidance activities; precrossing ROBO1 isoforms are inhibited to allow midline crossing, while postcrossing disinhibition prevents midline re-entry; this provides temporal regulation of Robo-mediated axonal repulsion independent of protein expression levels.","method":"Alternative splicing analysis; NOVA factor manipulation; in vitro Slit repulsion assay; commissural axon trajectory analysis in vivo","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — combination of splicing factor manipulation, isoform-specific functional assays, and in vivo guidance phenotype analysis","pmids":["31392959"],"is_preprint":false},{"year":2019,"finding":"Slit2/Robo1 signaling promotes tumor metastasis in colorectal carcinoma partly through activation of the TGF-β/Smads pathway; blocking Slit2-Robo1 binding inactivates TGF-β/Smads and inhibits tumor cell migration, which can be partially restored by exogenous TGF-β1.","method":"Robo1 blocking antibody; TGF-β1 rescue experiment; Smad phosphorylation Western blot; migration assay; in vivo tumor models","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor blockade with TGF-β1 rescue experiment establishing pathway ordering; single lab","pmids":["31242633"],"is_preprint":false},{"year":2020,"finding":"SLIT2 inhibits macropinocytosis in macrophages and RAS-transformed cancer cells through Robo1; mechanistically, SLIT2/ROBO1 signaling induces cytoskeletal changes that oppose cortical cytoskeletal remodeling required for macropinocytosis; in vivo, SLIT2 attenuates muramyl dipeptide uptake, preventing NOD2-dependent NF-κB activation and CXCL1 secretion.","method":"In vitro macropinocytosis assays; in vivo mouse model with SLIT2 treatment; blocking endogenous SLIT2; cytoskeletal imaging; NOD2/NF-κB pathway analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro and in vivo experiments with multiple mechanistic readouts (cytoskeletal, NF-κB, CXCL1); rigorous study","pmids":["32807784"],"is_preprint":false},{"year":2020,"finding":"PRRG4 promotes breast cancer metastasis by recruiting E3 ubiquitin ligase NEDD4 (through LPSY and PPPY motifs) to ubiquitinate and degrade Robo1, thereby activating Src and FAK signaling and promoting invasion.","method":"Co-immunoprecipitation; ubiquitination assay; PRRG4 knockdown/overexpression; experimental metastasis model; Western blot for Src/FAK phosphorylation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with domain mapping, ubiquitination assay establishing NEDD4 as E3 ligase for Robo1; single lab","pmids":["33037408"],"is_preprint":false},{"year":2010,"finding":"Src kinase activates Abl kinase, which stabilizes Robo1 protein to promote cell migration; inhibition of Abl by siRNA or kinase inhibitors decreases Robo1 protein levels and suppresses migration of transformed cells; Robo1 utilizes Cdc42 and Rac1 GTPases downstream to induce cell migration.","method":"Abl siRNA knockdown; pharmacological kinase inhibitors; Western blot for Robo1 protein levels; migration assay; small GTPase activation assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and pharmacological inhibition with defined mechanistic readouts (Robo1 stability, GTPase activity); single lab","pmids":["21301049"],"is_preprint":false},{"year":2012,"finding":"Slit2N/Robo1 inhibits HIV-gp120-induced migration and podosome formation in immature dendritic cells by causing Robo1 to sequester WASp and LSP1 away from β-actin, preventing WASp-Arp2/3-β-actin complex formation; Slit2N also inhibits Src signaling and downstream Rac1, Pyk2, paxillin, and CDC42 activation.","method":"Co-immunoprecipitation; transendothelial migration assay; podosome formation assay; Rac1/CDC42 activation assay; Src signaling Western blot","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating Robo1-WASp/LSP1 sequestration with functional migration and podosome readouts; single lab","pmids":["23119100"],"is_preprint":false},{"year":2015,"finding":"Slit2/Robo1 signaling is required for Drosophila Robo1 midline repulsion; Ig1 domain deletion disrupts Slit binding in vitro and abolishes ectopic midline repulsion in gain-of-function studies; Ig1 is not required for proper Robo1 expression, axonal localization, or Commissureless-dependent regulation in vivo.","method":"In vitro Slit binding assay in Drosophila cells; Drosophila in vivo gain-of-function and genetic rescue assays; immunostaining for axonal localization and Comm-dependent regulation","journal":"G3 (Bethesda, Md.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro binding assay combined with in vivo genetic rescue, domain deletion analysis establishing Ig1 as essential for Slit-dependent activity","pmids":["26362767"],"is_preprint":false},{"year":2018,"finding":"Fn3 domain deletion in Drosophila Robo1 impairs exclusion of Robo1 from commissures and reduces Commissureless-dependent downregulation of Robo1, while Fn1 and Fn2 deletions do not affect Slit binding, expression, midline repulsive function, or axonal localization; all three Fn repeats are individually dispensable for midline repulsion itself.","method":"In vivo domain deletion analysis (Fn1, Fn2, Fn3); in vitro Slit binding assay; midline repulsion genetic rescue assay; immunostaining for localization and Comm-dependent regulation","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion series with in vitro and in vivo functional assays, single lab","pmids":["29217730"],"is_preprint":false},{"year":2015,"finding":"Robo1 promotes angiogenesis in hepatocellular carcinoma by promoting Cdc42 expression in endothelial cells; knockdown of Robo1 distorts the actin cytoskeleton in HUVECs; in vivo, overexpression of Robo1 in endothelial cells mixed with hepatoma cells enhances tumor growth and microvessel density.","method":"Robo1 overexpression and knockdown in HUVECs; nude mouse tumor implantation; Cdc42 expression analysis; actin cytoskeleton imaging","journal":"Tumour biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo experiments with Cdc42 mechanistic readout; single lab","pmids":["26022159"],"is_preprint":false},{"year":2015,"finding":"Slit2/Robo1 signaling inhibits HUVEC migration in a Robo4-dependent manner; Robo4 negatively regulates the Slit2-Robo1 migratory signal through an intracellular mechanism; CdGAP (a Rac1/Cdc42-specific GTPase activating protein) was identified by targeted proteomics as a Robo1-interacting protein and co-immunoprecipitated with Robo1.","method":"Boyden chamber migration assay; Robo1 and Robo4 siRNA knockdown; Robo1 extracellular domain blocking; targeted proteomics; co-immunoprecipitation of Robo1-CdGAP","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP identifying CdGAP as Robo1 partner combined with functional migration assay; single lab","pmids":["26713366"],"is_preprint":false},{"year":2021,"finding":"YY1 transcription factor directly binds the ROBO1 promoter to promote transcription, as demonstrated by luciferase reporter, ChIP, and EMSA assays; ROBO1 inhibits pancreatic cancer cell proliferation through the CCNA2/CDK2 axis, causing S-phase arrest.","method":"Luciferase reporter gene assay; chromatin immunoprecipitation (ChIP); electrophoretic mobility shift assay (EMSA); cell cycle analysis; subcutaneous xenograft in nude mice","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — three orthogonal assays (luciferase, ChIP, EMSA) confirming YY1-ROBO1 promoter interaction, plus functional downstream pathway (CCNA2/CDK2); single lab but multiple rigorous methods","pmids":["33714986"],"is_preprint":false},{"year":2022,"finding":"Glial-secreted Netrins regulate Drosophila Robo1 signaling threshold levels in neural stem cells; Netrin-Frazzled/DCC signaling modulates Robo1 levels through Abelson kinase; unbalanced Robo1 signaling impairs asymmetric stem cell division by dysregulating aPKC, Canoe/Afadin, and Numb through Rac1 and Cdc42.","method":"Drosophila genetics (Netrin, Frazzled/DCC, Abelson kinase, Robo1 manipulations); immunostaining for ASCD machinery components; larval brain neuroblast lineage analysis","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in Drosophila model with defined downstream effectors (Rac1/Cdc42/aPKC axis); single lab","pmids":["35472309"],"is_preprint":false},{"year":2023,"finding":"ROBO1 exhibits dependence receptor (DR) characteristics in pancreatic ductal adenocarcinoma liver metastasis; PTP4A2 (protein tyrosine phosphatase 4A2) dephosphorylates ROBO1 in recurrent glioblastoma to modulate its downstream signaling and tumorigenicity; genetic or pharmacological inhibition of PTP4A2 acts through ROBO1; ROBO1-targeted CAR T cells eradicate tumors in xenograft models.","method":"CRISPR genetic perturbation screens; PTP4A2 inhibition; ROBO1 dephosphorylation assay; CDX xenograft models; CAR T cell therapy","journal":"Nature medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screens combined with mechanistic validation (dephosphorylation axis, in vivo CAR T efficacy); single study","pmids":["39095594"],"is_preprint":false},{"year":2023,"finding":"SLIT2-ROBO1-mediated coadaptation facilitates implantation and outgrowth of pancreatic ductal adenocarcinoma disseminated tumor cells in the liver premetastatic niche; ROBO1 displays dependence receptor characteristics; a ROBO1-neutralizing antibody significantly attenuates liver metastasis.","method":"Mouse premetastatic/macrometastatic niche models; CT/3D bioluminescence imaging; ROBO1 neutralizing antibody; human PDAC patient samples; mechanistic dependence receptor characterization","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo mouse models with antibody intervention and defined mechanistic characteristic (dependence receptor); single lab","pmids":["36792623"],"is_preprint":false}],"current_model":"ROBO1 is a transmembrane immunoglobulin superfamily receptor that binds Slit ligands (via its Ig1 domain, facilitated by heparan sulfate co-receptors binding at two distinct sites) and signals through intracellular interactions with effectors including srGAP1, DOCK1, CdGAP, and WASP-family proteins to regulate Cdc42/Rac1 GTPase activity, actin cytoskeletal remodeling, and cell migration; it forms homo- and hetero-dimeric (with Robo4) ectodomain assemblies and undergoes metalloproteinase/gamma-secretase sequential cleavage with nuclear translocation of its intracellular fragment; its expression is regulated by promoter methylation, ubiquitination via NEDD4 (counteracted by USP33 deubiquitylation), Abl kinase-mediated stabilization, post-transcriptional repression by multiple miRNAs (miR-218, miR-92, miR-203, miR-219), YY1 transcriptional activation, and alternative splicing of a conserved microexon; in vivo, Robo1 is essential for midline axon guidance, cortical interneuron migration, forebrain commissure formation, neocortical progenitor proliferation, lung development, and mammary branching morphogenesis, and its downstream pathways include WNT/β-catenin, TGF-β/Smad, PI3K/AKT, Src/ERK, and PKA/DCC signaling axes depending on cellular context."},"narrative":{"mechanistic_narrative":"ROBO1 is a transmembrane immunoglobulin-superfamily receptor for secreted Slit ligands that governs cell migration, axon guidance, and tissue morphogenesis by transducing repulsive cues into actin cytoskeletal reorganization [PMID:16690755, PMID:26362767]. Ligand engagement requires the first Ig domain, which is essential for Slit binding and for repulsive activity in vivo [PMID:26362767], and is potentiated by heparan/heparin co-receptors that bind Robo1 at two distinct sites — a previously known site and a second within the disordered N-terminus — with 6-O-sulfation being critical for high-affinity recognition [PMID:23994753, PMID:25752613, PMID:27611601]. Structurally, the Robo1 ectodomain assembles into compact dimers and back-to-back tetramers via central Ig domains, with Slit2-N binding inducing a conformational change rather than altering oligomeric state, and Robo1 also forms heterodimers with Robo4 [PMID:29307485, PMID:18948384]. Intracellularly, Robo1 controls the Cdc42/Rac1 GTPase switch through partners including srGAP1, DOCK1, CdGAP, and WASP-family proteins to direct actin remodeling, lamellipodia/filopodia formation, focal-adhesion and cell-tension signaling [PMID:27923383, PMID:24752651, PMID:26713366, PMID:18948384, PMID:26975850]. In neural development Robo1 prevents post-crossing commissural axon stalling and midline re-entry, restrains cortical interneuron migration, and limits neocortical progenitor proliferation, with its repulsive output temporally tuned by miR-92 translational repression and NOVA-regulated microexon splicing rather than by expression level alone [PMID:16690755, PMID:20631173, PMID:24741061, PMID:30184503, PMID:31392959]. Beyond the nervous system Robo1 directs lung maturation, mammary stem-cell self-renewal and branching via WNT/β-catenin and SNAI1/mINSC control, retinal and tumor angiogenesis through Rac1 and VEGFR2/ERK, and immune-cell migration and macropinocytosis [PMID:11734623, PMID:21664580, PMID:26440891, PMID:25894826, PMID:32807784]. In cancer Robo1 behaves as a context-dependent tumor suppressor silenced by promoter hypermethylation and destabilized by NEDD4-mediated ubiquitination opposed by USP33 deubiquitylation, and its protein level and signaling are further set by Abl-mediated stabilization, PTP4A2 dephosphorylation, and YY1 transcriptional activation [PMID:15374951, PMID:12082532, PMID:33037408, PMID:30896071, PMID:21301049, PMID:39095594, PMID:33714986]. Human ROBO1 also undergoes sequential metalloproteinase and gamma-secretase cleavage producing an intracellular fragment that accumulates in the nucleus of cancer cells [PMID:20471383].","teleology":[{"year":2001,"claim":"Established that Robo1 has an essential developmental function beyond neural tissue, demonstrating it is required for lung maturation and epithelial homeostasis.","evidence":"Targeted exon-5 (Ig1) knockout in mice with histopathology","pmids":["11734623"],"confidence":"High","gaps":["Did not define the Slit ligand or signaling effectors in lung","Mechanism linking receptor loss to delayed alveolar maturation unresolved"]},{"year":2002,"claim":"Identified an epigenetic inactivation mechanism, showing ROBO1 is silenced by promoter CpG-island hypermethylation in multiple carcinomas and reactivated by demethylation.","evidence":"Bisulfite sequencing, MSP, and 5-aza-dC reactivation in tumor lines","pmids":["12082532"],"confidence":"High","gaps":["Did not establish downstream tumor-suppressive signaling","Causal contribution to tumor initiation not shown in this study"]},{"year":2004,"claim":"Provided in vivo genetic evidence that Robo1 acts as a two-hit tumor suppressor, with the retained allele silenced by methylation in spontaneous tumors.","evidence":"Heterozygous knockout mice with LOH, methylation, and protein analyses","pmids":["15374951"],"confidence":"High","gaps":["Tumor-suppressive effector pathway not defined","Tissue-specificity of suppression unexplained"]},{"year":2006,"claim":"Defined Robo1's dual neural role, showing it both shapes forebrain commissural axon tracts and restrains cortical interneuron migration.","evidence":"Robo1 knockout mice with DTI-MRI, immunohistochemistry, cell counting","pmids":["16690755"],"confidence":"High","gaps":["Did not separate cell-autonomous from non-autonomous effects","Intracellular signaling not resolved"]},{"year":2007,"claim":"Connected receptor surface availability to migration outcome, showing a kinesin/Trio-GEF complex controls SAX-3/Robo1 surface levels and physically engages the intracellular domain.","evidence":"C. elegans epistasis, co-IP, and receptor abundance measurement","pmids":["17237778"],"confidence":"High","gaps":["Mammalian relevance of VAB-8L/UNC-73 mechanism not tested here","Direct effect on GTPase output not measured"]},{"year":2007,"claim":"Demonstrated that Robo1 is the principal functional receptor mediating Slit2 chemorepulsion in tumor cells.","evidence":"siRNA knockdown with Boyden-chamber migration in glioma lines","pmids":["17968499"],"confidence":"Medium","gaps":["Single method, single cell type","Downstream effectors not identified in this study"]},{"year":2008,"claim":"Linked Robo1 to actin-nucleation machinery, showing it heterodimerizes with Robo4 and that the receptor pair connects to WASP/N-WASP/WIP for endothelial filopodia.","evidence":"Y2H, GST pulldown, siRNA, co-IP, endothelial assays","pmids":["18948384"],"confidence":"High","gaps":["WASP-family binding mapped to Robo4 ICD, not Robo1 directly","In vivo requirement not tested"]},{"year":2010,"claim":"Resolved how the Robo paralogs divide labor at the midline, with Robo1 specifically preventing post-crossing axon stalling and midline re-entry.","evidence":"Robo1/2/3 combinatorial knockout mice, spinal axon tracing","pmids":["20631173"],"confidence":"High","gaps":["Molecular basis of paralog-specific output not defined","Ligand-specificity at the midline not dissected here"]},{"year":2010,"claim":"Showed human ROBO1 is processed by regulated intramembrane proteolysis, generating a nuclear-accumulating intracellular fragment in cancer cells.","evidence":"Cleavage-fragment Western blots, TAPI-1/L-685,458 inhibition, nuclear fractionation","pmids":["20471383"],"confidence":"Medium","gaps":["Nuclear function of the ICD fragment not determined","Single-lab biochemistry without in vivo confirmation"]},{"year":2010,"claim":"Established post-translational control of Robo1 abundance, with Src-activated Abl kinase stabilizing the protein to drive migration via Cdc42/Rac1.","evidence":"Abl siRNA/inhibitors, Robo1 Western blots, GTPase activation assays","pmids":["21301049"],"confidence":"Medium","gaps":["Direct phosphorylation site not mapped","Single-lab pharmacological evidence"]},{"year":2011,"claim":"Placed Robo1 in a TGF-β-to-WNT regulatory axis controlling tissue branching, showing it restricts mammary basal-cell proliferation by limiting nuclear β-catenin.","evidence":"Conditional knockout mice, whole-mount and β-catenin immunofluorescence, TGF-β1 treatment","pmids":["21664580"],"confidence":"High","gaps":["Mechanistic link from receptor to β-catenin partitioning not fully resolved","Direct effectors downstream of Robo1 not identified here"]},{"year":2012,"claim":"Defined a sequestration mechanism, showing Slit2/Robo1 captures WASp and LSP1 away from β-actin to block podosome formation and migration.","evidence":"Co-IP, transendothelial migration, podosome and Rac1/CDC42 assays in dendritic cells","pmids":["23119100"],"confidence":"Medium","gaps":["Direct vs indirect Robo1-WASp binding not distinguished","Single-lab study"]},{"year":2013,"claim":"Began mapping the heparin-binding biochemistry of Robo1, quantifying affinity and the sulfation groups required for co-receptor engagement.","evidence":"SPR with systematically modified heparin","pmids":["23994753"],"confidence":"Medium","gaps":["Binding-site location not yet mapped","Functional consequence for Slit signaling not tested here"]},{"year":2013,"claim":"Provided anti-invasive mechanism in tumor cells, linking Slit2-Robo1 to suppression of Cdc42 activity.","evidence":"Cdc42 pull-down, siRNA, invasion assay in glioma","pmids":["21113198"],"confidence":"Low","gaps":["Single GTPase assay without independent replication","Abstract-level mechanistic detail only","Direct GAP/GEF effector not identified"]},{"year":2014,"claim":"Identified DOCK1 as a Robo1 intracellular effector controlling Rac1 and cadherin-based adhesion, explaining anti-migratory tumor-suppressor activity.","evidence":"Domain-mapping mutants, co-IP, confocal, invasion assays in prostate cancer","pmids":["24752651"],"confidence":"Medium","gaps":["In vivo relevance not tested","Single-lab co-IP without reciprocal validation"]},{"year":2014,"claim":"Revealed cross-talk between guidance systems, showing FLRT3 acts as a Robo1 co-receptor allowing Slit1 to switch on Netrin attraction via PKA/DCC.","evidence":"Co-receptor identification, axon guidance and PKA/surface-DCC assays, knockout mice","pmids":["24560577"],"confidence":"High","gaps":["Structural basis of Robo1-FLRT3 association not resolved","Generality beyond thalamic axons unknown"]},{"year":2014,"claim":"Established a proliferative-control role in cortical development, showing Robo1 loss prolongs progenitor proliferation and expands neuron output.","evidence":"Knockout mice, in utero shRNA electroporation, BrdU/Ki67 analysis","pmids":["24741061"],"confidence":"High","gaps":["Signaling pathway coupling Robo1 to progenitor cell cycle not defined","Ligand source in vivo not identified"]},{"year":2015,"claim":"Demonstrated a pro-angiogenic role, showing endothelial Robo1/Robo2 are required for Slit2- and VEGF-induced Rac1 activation and retinal vessel growth.","evidence":"Endothelial conditional knockouts, retinal angiogenesis and Rac1/lamellipodia assays","pmids":["25894826"],"confidence":"High","gaps":["Mechanism of VEGF/Robo cross-talk not fully resolved","Direct Rac GEF not identified here"]},{"year":2015,"claim":"Identified srGAP1 as the Robo1 effector mediating anti-migratory Cdc42 inhibition in cancer and immune-cell contexts.","evidence":"Co-IP, immunofluorescence, Cdc42 pull-down, migration and brain-injury models","pmids":["27923383","26550694"],"confidence":"Medium","gaps":["Reciprocal validation of the interaction limited","Single-lab co-IP evidence"]},{"year":2015,"claim":"Showed Robo1 governs stem-cell division mode, controlling asymmetric vs symmetric divisions through SNAI1/mINSC and spindle orientation.","evidence":"Conditional knockout, single-cell lineage tracing, SLIT2 rescue","pmids":["26440891"],"confidence":"High","gaps":["Direct molecular link from receptor to SNAI1 not defined","Generality to other stem-cell systems untested"]},{"year":2015,"claim":"Documented context-dependent pro-fibrotic and pro-oncogenic signaling, with Robo1 activating Smad2/3, PI3K/AKT, and Src/Wnt/ERK axes.","evidence":"Transgenic/knockout mice, neutralizing antibody, pathway Western blots in liver, intestine, and angiogenesis models","pmids":["26264936","25605242","26244297","26022159"],"confidence":"Medium","gaps":["Apparent opposite (tumor-promoting vs suppressing) roles not reconciled mechanistically","Direct vs indirect pathway activation unclear"]},{"year":2015,"claim":"Expanded the Robo1 effector network, identifying CdGAP as a partner and Robo4 as a negative modulator of the Slit2-Robo1 migratory signal.","evidence":"Targeted proteomics, co-IP, Robo1/Robo4 siRNA migration assays","pmids":["26713366"],"confidence":"Medium","gaps":["CdGAP interaction not reciprocally validated","In vivo relevance untested"]},{"year":2015,"claim":"Validated the Ig1 domain as the obligatory Slit-binding and repulsion module while showing it is dispensable for receptor localization and Comm regulation.","evidence":"Drosophila in vitro Slit binding and in vivo gain-of-function/rescue with Ig1 deletion","pmids":["26362767"],"confidence":"High","gaps":["Does not address co-receptor contributions to binding","Mammalian Ig1 in vivo requirement inferred, not shown here"]},{"year":2016,"claim":"Resolved the structural basis of Robo1-heparan sulfate recognition, defining two binding sites, the critical 6-O-sulfate requirement, and a synthetic ligand that reverses Slit2-Robo1 migration inhibition.","evidence":"Hydroxyl-radical footprinting, NMR (STD/trNOE), SPR, HS synthesis, mutagenesis, migration assay","pmids":["25752613","27611601","27653286"],"confidence":"High","gaps":["Functional role of N-terminal HS site in vivo not established","How HS binding alters Slit-dependent signaling kinetics unresolved"]},{"year":2016,"claim":"Tied mechanical ECM cues to Robo1 expression and cytoskeletal tension, showing matrix stiffness raises Robo1 via miR-203 to drive Rac/FAK-dependent contractility.","evidence":"miR-203/Robo1 manipulation, Rac/FAK assays, conditional knockout, tunable hydrogels","pmids":["26975850"],"confidence":"Medium","gaps":["Direct miR-203/Robo1 targeting versus indirect regulation not fully separated","Single-lab study"]},{"year":2018,"claim":"Defined the ectodomain architecture, showing Robo1 forms dimers and back-to-back tetramers and that Slit2-N triggers conformation rather than oligomerization change.","evidence":"X-ray crystallography of Ig1-4/Ig5 and negative-stain EM with Slit2-N imaging","pmids":["29307485"],"confidence":"High","gaps":["Functional consequence of the conformational change for signaling not shown","Full-length receptor in membrane not visualized"]},{"year":2018,"claim":"Identified Robo1 ubiquitination control, showing USP33 deubiquitinates and stabilizes Robo1, with its loss reducing Slit2-Robo1 tumor-suppressive signaling.","evidence":"Co-IP, ubiquitination assay, siRNA, migration/EMT models in gastric cancer","pmids":["30896071"],"confidence":"Medium","gaps":["Ubiquitination sites not mapped","Single-lab evidence"]},{"year":2018,"claim":"Linked Robo1 to tumor metabolism, showing SLIT2/ROBO1 drives the Warburg effect via SRC/ERK/c-MYC/PFKFB2.","evidence":"Knockdown/overexpression, xenografts, pathway Western blots, metabolic assays","pmids":["29523788"],"confidence":"Medium","gaps":["Direct receptor-to-SRC coupling not demonstrated","Single-lab study"]},{"year":2018,"claim":"Established translational, temporal control of Robo1 in axon guidance, showing miR-92 represses Robo1 translation to delay Slit2 sensitivity until after midline crossing.","evidence":"miR-92 gain/loss, 3'UTR luciferase reporter, in vitro repulsion, in vivo chick axon labeling","pmids":["30184503"],"confidence":"High","gaps":["Upstream control of miR-92 timing not defined","Mammalian conservation of timing not tested here"]},{"year":2019,"claim":"Revealed a second layer of temporal regulation, showing NOVA-controlled microexon splicing generates Robo1 isoforms with distinct repulsive activities independent of expression level.","evidence":"Splicing analysis, NOVA manipulation, isoform repulsion assays, in vivo axon trajectories","pmids":["31392959"],"confidence":"High","gaps":["Structural basis of isoform activity difference unresolved","Regulation of NOVA timing in vivo not defined"]},{"year":2019,"claim":"Showed a pro-metastatic signaling output in carcinoma, with Slit2-Robo1 activating TGF-β/Smad to drive migration.","evidence":"Robo1 blocking antibody, TGF-β1 rescue, Smad phosphorylation, in vivo tumor models","pmids":["31242633"],"confidence":"Medium","gaps":["Direct receptor-to-Smad coupling not shown","Reconciliation with tumor-suppressor role absent"]},{"year":2020,"claim":"Extended Robo1 function to innate immunity, showing SLIT2/ROBO1 blocks macropinocytosis and downstream NOD2/NF-κB inflammatory signaling by opposing cortical cytoskeletal remodeling.","evidence":"Macropinocytosis assays, cytoskeletal imaging, in vivo SLIT2 treatment, NF-κB/CXCL1 analysis","pmids":["32807784"],"confidence":"High","gaps":["Cytoskeletal effectors linking Robo1 to macropinosome closure not fully identified"]},{"year":2020,"claim":"Identified an E3-ligase axis controlling Robo1 levels, with PRRG4 recruiting NEDD4 to ubiquitinate and degrade Robo1, de-repressing Src/FAK and invasion.","evidence":"Co-IP with motif mapping, ubiquitination assay, metastasis model, Src/FAK Western blots","pmids":["33037408"],"confidence":"Medium","gaps":["NEDD4 ubiquitination sites on Robo1 not mapped","Single-lab study"]},{"year":2021,"claim":"Defined transcriptional activation and a cell-cycle effector arm, showing YY1 directly drives ROBO1 transcription and ROBO1 arrests pancreatic cancer cells via the CCNA2/CDK2 axis.","evidence":"Luciferase, ChIP, EMSA, cell-cycle analysis, xenografts","pmids":["33714986"],"confidence":"High","gaps":["Link from receptor signaling to CCNA2/CDK2 regulation not mechanistically traced","Generality beyond pancreatic cancer untested"]},{"year":2022,"claim":"Showed Robo1 signaling threshold sets stem-cell division asymmetry, with glial Netrin-Frazzled/DCC tuning Robo1 via Abelson kinase to control aPKC/Canoe/Numb through Rac1/Cdc42.","evidence":"Drosophila genetics and ASCD-component immunostaining in neuroblasts","pmids":["35472309"],"confidence":"Medium","gaps":["Mammalian conservation untested","Direct molecular link from Robo1 level to polarity machinery unresolved"]},{"year":2023,"claim":"Characterized Robo1 as a dependence receptor and actionable target in pancreatic and brain cancer, with PTP4A2 dephosphorylating ROBO1 and ROBO1-directed antibodies/CAR-T attenuating metastasis and tumor growth.","evidence":"CRISPR screens, PTP4A2 inhibition, dephosphorylation assays, premetastatic-niche models, neutralizing antibody and CAR-T xenografts","pmids":["39095594","36792623"],"confidence":"Medium","gaps":["Dependence-receptor death signaling mechanism not fully defined","PTP4A2 dephosphorylation sites on ROBO1 not mapped"]},{"year":null,"claim":"How a single Slit-Robo1 receptor produces context-opposite outcomes — tumor suppression versus promotion, repulsion versus attraction, proliferation restraint versus angiogenesis — through differential effector engagement, proteolysis, and isoform usage remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking effector choice to opposite cellular outputs","Nuclear function of the cleaved ICD fragment unknown","In vivo significance of ectodomain tetramerization and dual HS sites untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[36,4,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[20,13,39,34]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[6,25,35]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[11,16,23,24]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,5,26]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[6,25,38]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,3,7,14,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,18,19,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,2,42,43]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[32,35,21]}],"complexes":[],"partners":["SLIT2","ROBO4","SRGAP1","DOCK1","CDGAP","USP33","NEDD4","FLRT3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y6N7","full_name":"Roundabout homolog 1","aliases":["Deleted in U twenty twenty","H-Robo-1"],"length_aa":1651,"mass_kda":180.9,"function":"Receptor for SLIT1 and SLIT2 that mediates cellular responses to molecular guidance cues in cellular migration, including axonal navigation at the ventral midline of the neural tube and projection of axons to different regions during neuronal development (PubMed:10102268, PubMed:24560577). Interaction with the intracellular domain of FLRT3 mediates axon attraction towards cells expressing NTN1 (PubMed:24560577). In axon growth cones, the silencing of the attractive effect of NTN1 by SLIT2 may require the formation of a ROBO1-DCC complex (By similarity). Plays a role in the regulation of cell migration via its interaction with MYO9B; inhibits MYO9B-mediated stimulation of RHOA GTPase activity, and thereby leads to increased levels of active, GTP-bound RHOA (PubMed:26529257). May be required for lung development (By similarity)","subcellular_location":"Cell membrane; Cell projection, axon; Endoplasmic reticulum-Golgi intermediate compartment membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y6N7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ROBO1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ROBO1","total_profiled":1310},"omim":[{"mim_id":"621003","title":"TRANSCRIPTION FACTOR Sp9; SP9","url":"https://www.omim.org/entry/621003"},{"mim_id":"620305","title":"NEUROOCULORENAL SYNDROME; NORS","url":"https://www.omim.org/entry/620305"},{"mim_id":"620303","title":"PITUITARY HORMONE DEFICIENCY, COMBINED OR ISOLATED, 8; CPHD8","url":"https://www.omim.org/entry/620303"},{"mim_id":"619266","title":"TRIM52 ANTISENSE RNA 1, NONCODING; TRIM52AS1","url":"https://www.omim.org/entry/619266"},{"mim_id":"618687","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH SHORT STATURE AND BEHAVIORAL ABNORMALITIES; IDDSSBA","url":"https://www.omim.org/entry/618687"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ROBO1"},"hgnc":{"alias_symbol":["DUTT1","FLJ21882","SAX3"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6N7","domains":[{"cath_id":"2.60.40.10","chopping":"67-166","consensus_level":"high","plddt":90.6731,"start":67,"end":166},{"cath_id":"2.60.40.10","chopping":"168-259","consensus_level":"medium","plddt":91.779,"start":168,"end":259},{"cath_id":"2.60.40.10","chopping":"268-344","consensus_level":"medium","plddt":91.6288,"start":268,"end":344},{"cath_id":"2.60.40.10","chopping":"357-451","consensus_level":"medium","plddt":85.6779,"start":357,"end":451},{"cath_id":"2.60.40.10","chopping":"453-550","consensus_level":"medium","plddt":85.7303,"start":453,"end":550},{"cath_id":"2.60.40.10","chopping":"558-658","consensus_level":"medium","plddt":88.2236,"start":558,"end":658},{"cath_id":"2.60.40.10","chopping":"669-772","consensus_level":"medium","plddt":87.438,"start":669,"end":772},{"cath_id":"2.60.40.10","chopping":"784-883","consensus_level":"high","plddt":83.5643,"start":784,"end":883}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6N7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6N7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6N7-F1-predicted_aligned_error_v6.png","plddt_mean":60.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ROBO1","jax_strain_url":"https://www.jax.org/strain/search?query=ROBO1"},"sequence":{"accession":"Q9Y6N7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6N7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6N7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6N7"}},"corpus_meta":[{"pmid":"20300657","id":"PMC_20300657","title":"MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor.","date":"2010","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/20300657","citation_count":379,"is_preprint":false},{"pmid":"21385904","id":"PMC_21385904","title":"MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway.","date":"2011","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/21385904","citation_count":243,"is_preprint":false},{"pmid":"16254601","id":"PMC_16254601","title":"The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia.","date":"2005","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16254601","citation_count":235,"is_preprint":false},{"pmid":"16690755","id":"PMC_16690755","title":"Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain.","date":"2006","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/16690755","citation_count":211,"is_preprint":false},{"pmid":"25894826","id":"PMC_25894826","title":"Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization.","date":"2015","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25894826","citation_count":159,"is_preprint":false},{"pmid":"11734623","id":"PMC_11734623","title":"Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene.","date":"2001","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/11734623","citation_count":130,"is_preprint":false},{"pmid":"17968499","id":"PMC_17968499","title":"Slit2 involvement in glioma cell migration is mediated by Robo1 receptor.","date":"2007","source":"Journal of neuro-oncology","url":"https://pubmed.ncbi.nlm.nih.gov/17968499","citation_count":105,"is_preprint":false},{"pmid":"18948384","id":"PMC_18948384","title":"Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors.","date":"2008","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/18948384","citation_count":101,"is_preprint":false},{"pmid":"12082532","id":"PMC_12082532","title":"Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers.","date":"2002","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/12082532","citation_count":98,"is_preprint":false},{"pmid":"20631173","id":"PMC_20631173","title":"Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance.","date":"2010","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/20631173","citation_count":98,"is_preprint":false},{"pmid":"21664580","id":"PMC_21664580","title":"SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number.","date":"2011","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/21664580","citation_count":88,"is_preprint":false},{"pmid":"16740745","id":"PMC_16740745","title":"Identification of ROBO1 as a novel hepatocellular carcinoma antigen and a potential therapeutic and diagnostic target.","date":"2006","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/16740745","citation_count":87,"is_preprint":false},{"pmid":"26264936","id":"PMC_26264936","title":"Activation of Slit2-Robo1 signaling promotes liver fibrosis.","date":"2015","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/26264936","citation_count":82,"is_preprint":false},{"pmid":"29523788","id":"PMC_29523788","title":"SLIT2/ROBO1 axis contributes to the Warburg effect in osteosarcoma through activation of SRC/ERK/c-MYC/PFKFB2 pathway.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29523788","citation_count":82,"is_preprint":false},{"pmid":"17237778","id":"PMC_17237778","title":"C. elegans VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell and growth-cone migrations.","date":"2007","source":"Nature neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/17237778","citation_count":78,"is_preprint":false},{"pmid":"17671114","id":"PMC_17671114","title":"Frequent inactivation of RAMP2, EFEMP1 and Dutt1 in lung cancer by promoter hypermethylation.","date":"2007","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/17671114","citation_count":74,"is_preprint":false},{"pmid":"24560577","id":"PMC_24560577","title":"FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.","date":"2014","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/24560577","citation_count":70,"is_preprint":false},{"pmid":"16685377","id":"PMC_16685377","title":"Robo1/Robo4: differential expression of angiogenic markers in colorectal cancer.","date":"2006","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/16685377","citation_count":66,"is_preprint":false},{"pmid":"25030704","id":"PMC_25030704","title":"Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25030704","citation_count":63,"is_preprint":false},{"pmid":"9608531","id":"PMC_9608531","title":"The DUTT1 gene, a novel NCAM family member is expressed in developing murine neural tissues and has an unusually broad pattern of expression.","date":"1998","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/9608531","citation_count":57,"is_preprint":false},{"pmid":"24167656","id":"PMC_24167656","title":"MicroRNA 203 Modulates Glioma Cell Migration via Robo1/ERK/MMP-9 Signaling.","date":"2013","source":"Genes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/24167656","citation_count":51,"is_preprint":false},{"pmid":"25752613","id":"PMC_25752613","title":"High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25752613","citation_count":51,"is_preprint":false},{"pmid":"20471383","id":"PMC_20471383","title":"Human ROBO1 is cleaved by metalloproteinases and gamma-secretase and migrates to the nucleus in cancer cells.","date":"2010","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/20471383","citation_count":50,"is_preprint":false},{"pmid":"15374951","id":"PMC_15374951","title":"Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.","date":"2004","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/15374951","citation_count":48,"is_preprint":false},{"pmid":"21113198","id":"PMC_21113198","title":"Slit2/Robo1 signaling in glioma migration and invasion.","date":"2010","source":"Neuroscience bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/21113198","citation_count":48,"is_preprint":false},{"pmid":"24741061","id":"PMC_24741061","title":"Robo1 modulates proliferation and neurogenesis in the developing neocortex.","date":"2014","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/24741061","citation_count":48,"is_preprint":false},{"pmid":"28323002","id":"PMC_28323002","title":"miR-218 inhibited tumor angiogenesis by targeting ROBO1 in gastric cancer.","date":"2017","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/28323002","citation_count":48,"is_preprint":false},{"pmid":"30896071","id":"PMC_30896071","title":"Reduced USP33 expression in gastric cancer decreases inhibitory effects of Slit2-Robo1 signalling on cell migration and EMT.","date":"2019","source":"Cell proliferation","url":"https://pubmed.ncbi.nlm.nih.gov/30896071","citation_count":46,"is_preprint":false},{"pmid":"32807784","id":"PMC_32807784","title":"SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32807784","citation_count":44,"is_preprint":false},{"pmid":"26440891","id":"PMC_26440891","title":"Mammary Stem Cell Self-Renewal Is Regulated by Slit2/Robo1 Signaling through SNAI1 and mINSC.","date":"2015","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/26440891","citation_count":44,"is_preprint":false},{"pmid":"21060114","id":"PMC_21060114","title":"The corticofugal neuron-associated genes ROBO1, SRGAP1, and CTIP2 exhibit an anterior to posterior gradient of expression in early fetal human neocortex development.","date":"2010","source":"Cerebral cortex (New York, N.Y. : 1991)","url":"https://pubmed.ncbi.nlm.nih.gov/21060114","citation_count":44,"is_preprint":false},{"pmid":"29743814","id":"PMC_29743814","title":"MicroRNA-365 promotes lung carcinogenesis by downregulating the USP33/SLIT2/ROBO1 signalling pathway.","date":"2018","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/29743814","citation_count":41,"is_preprint":false},{"pmid":"25786906","id":"PMC_25786906","title":"SLIT2/ROBO1-miR-218-1-RET/PLAG1: a new disease pathway involved in Hirschsprung's disease.","date":"2015","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25786906","citation_count":40,"is_preprint":false},{"pmid":"14689480","id":"PMC_14689480","title":"Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system.","date":"2004","source":"The Journal of comparative neurology","url":"https://pubmed.ncbi.nlm.nih.gov/14689480","citation_count":40,"is_preprint":false},{"pmid":"27611601","id":"PMC_27611601","title":"Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.","date":"2016","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/27611601","citation_count":40,"is_preprint":false},{"pmid":"22262894","id":"PMC_22262894","title":"Human ROBO1 regulates interaural interaction in auditory pathways.","date":"2012","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/22262894","citation_count":40,"is_preprint":false},{"pmid":"25605242","id":"PMC_25605242","title":"Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/25605242","citation_count":39,"is_preprint":false},{"pmid":"26550694","id":"PMC_26550694","title":"Recombinant Slit2 attenuates neuroinflammation after surgical brain injury by inhibiting peripheral immune cell infiltration via Robo1-srGAP1 pathway in a rat model.","date":"2015","source":"Neurobiology of disease","url":"https://pubmed.ncbi.nlm.nih.gov/26550694","citation_count":37,"is_preprint":false},{"pmid":"26244297","id":"PMC_26244297","title":"Slit2 Promotes Angiogenic Activity Via the Robo1-VEGFR2-ERK1/2 Pathway in Both In Vivo and In Vitro Studies.","date":"2015","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/26244297","citation_count":36,"is_preprint":false},{"pmid":"29299781","id":"PMC_29299781","title":"Slit2/Robo1 signaling is involved in angiogenesis of glomerular endothelial cells exposed to a diabetic-like environment.","date":"2018","source":"Angiogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/29299781","citation_count":36,"is_preprint":false},{"pmid":"23733161","id":"PMC_23733161","title":"The microRNA-218 and ROBO-1 signaling axis correlates with the lymphatic metastasis of pancreatic cancer.","date":"2013","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/23733161","citation_count":36,"is_preprint":false},{"pmid":"25010661","id":"PMC_25010661","title":"MicroRNA-218 inhibits cell invasion and migration of pancreatic cancer via regulating ROBO1.","date":"2014","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/25010661","citation_count":35,"is_preprint":false},{"pmid":"28402530","id":"PMC_28402530","title":"Mutations in the Human ROBO1 Gene in Pituitary Stalk Interruption Syndrome.","date":"2017","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/28402530","citation_count":35,"is_preprint":false},{"pmid":"24752651","id":"PMC_24752651","title":"ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: study in African-American and Caucasian prostate cancer models.","date":"2014","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/24752651","citation_count":35,"is_preprint":false},{"pmid":"26975850","id":"PMC_26975850","title":"Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness.","date":"2016","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26975850","citation_count":34,"is_preprint":false},{"pmid":"22898079","id":"PMC_22898079","title":"Silencing of miRNA-218 promotes migration and invasion of breast cancer via Slit2-Robo1 pathway.","date":"2012","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/22898079","citation_count":34,"is_preprint":false},{"pmid":"32398956","id":"PMC_32398956","title":"Slit2/Robo1 Mitigates DSS-induced Ulcerative Colitis by Activating Autophagy in Intestinal Stem Cell.","date":"2020","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32398956","citation_count":33,"is_preprint":false},{"pmid":"12123796","id":"PMC_12123796","title":"Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development.","date":"2002","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12123796","citation_count":31,"is_preprint":false},{"pmid":"27923383","id":"PMC_27923383","title":"srGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer.","date":"2016","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/27923383","citation_count":31,"is_preprint":false},{"pmid":"25755718","id":"PMC_25755718","title":"miR-1179 promotes cell invasion through SLIT2/ROBO1 axis in esophageal squamous cell carcinoma.","date":"2015","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/25755718","citation_count":30,"is_preprint":false},{"pmid":"28924964","id":"PMC_28924964","title":"MiR-490-5p Inhibits Hepatocellular Carcinoma Cell Proliferation, Migration and Invasion by Directly Regulating ROBO1.","date":"2017","source":"Pathology oncology research : POR","url":"https://pubmed.ncbi.nlm.nih.gov/28924964","citation_count":30,"is_preprint":false},{"pmid":"28592524","id":"PMC_28592524","title":"Loss of function in ROBO1 is associated with tetralogy of Fallot and septal defects.","date":"2017","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28592524","citation_count":30,"is_preprint":false},{"pmid":"36792623","id":"PMC_36792623","title":"Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36792623","citation_count":29,"is_preprint":false},{"pmid":"29307485","id":"PMC_29307485","title":"Robo1 Forms a Compact Dimer-of-Dimers Assembly.","date":"2018","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/29307485","citation_count":29,"is_preprint":false},{"pmid":"30184503","id":"PMC_30184503","title":"miR-92 Suppresses Robo1 Translation to Modulate Slit Sensitivity in Commissural Axon Guidance.","date":"2018","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/30184503","citation_count":29,"is_preprint":false},{"pmid":"21998696","id":"PMC_21998696","title":"Influence of ROBO1 and RORA on risk of age-related macular degeneration reveals genetically distinct phenotypes in disease pathophysiology.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21998696","citation_count":29,"is_preprint":false},{"pmid":"31392959","id":"PMC_31392959","title":"Temporal regulation of axonal repulsion by alternative splicing of a conserved microexon in mammalian Robo1 and Robo2.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/31392959","citation_count":29,"is_preprint":false},{"pmid":"39095594","id":"PMC_39095594","title":"Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.","date":"2024","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39095594","citation_count":28,"is_preprint":false},{"pmid":"24612512","id":"PMC_24612512","title":"Association of the ROBO1 gene with reading disabilities in a family-based analysis.","date":"2014","source":"Genes, brain, and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/24612512","citation_count":28,"is_preprint":false},{"pmid":"25789001","id":"PMC_25789001","title":"miR-218 inhibits the migration and invasion of glioma U87 cells through the Slit2-Robo1 pathway.","date":"2015","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/25789001","citation_count":28,"is_preprint":false},{"pmid":"26081620","id":"PMC_26081620","title":"MicroRNA-219-5p exerts tumor suppressor function by targeting ROBO1 in glioblastoma.","date":"2015","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26081620","citation_count":28,"is_preprint":false},{"pmid":"33236535","id":"PMC_33236535","title":"Crosstalk between the activated Slit2-Robo1 pathway and TGF-β1 signalling promotes cardiac fibrosis.","date":"2020","source":"ESC heart failure","url":"https://pubmed.ncbi.nlm.nih.gov/33236535","citation_count":27,"is_preprint":false},{"pmid":"23994753","id":"PMC_23994753","title":"Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans.","date":"2013","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/23994753","citation_count":27,"is_preprint":false},{"pmid":"31719262","id":"PMC_31719262","title":"Dynamic expression of Slit1-3 and Robo1-2 in the mouse peripheral nervous system after injury.","date":"2020","source":"Neural regeneration research","url":"https://pubmed.ncbi.nlm.nih.gov/31719262","citation_count":27,"is_preprint":false},{"pmid":"33037408","id":"PMC_33037408","title":"PRRG4 promotes breast cancer metastasis through the recruitment of NEDD4 and downregulation of Robo1.","date":"2020","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/33037408","citation_count":27,"is_preprint":false},{"pmid":"26022159","id":"PMC_26022159","title":"Robo1 promotes angiogenesis in hepatocellular carcinoma through the Rho family of guanosine triphosphatases' signaling pathway.","date":"2015","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26022159","citation_count":26,"is_preprint":false},{"pmid":"32922520","id":"PMC_32922520","title":"Long non-coding RNA LINC00473 acts as a microRNA-29a-3p sponge to promote hepatocellular carcinoma development by activating Robo1-dependent PI3K/AKT/mTOR signaling pathway.","date":"2020","source":"Therapeutic advances in medical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32922520","citation_count":26,"is_preprint":false},{"pmid":"35838343","id":"PMC_35838343","title":"Slit2/Robo1 signaling inhibits small-cell lung cancer by targeting β-catenin signaling in tumor cells and macrophages.","date":"2023","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35838343","citation_count":25,"is_preprint":false},{"pmid":"36277474","id":"PMC_36277474","title":"Tumor-Derived Exosomal miR-29b Reduces Angiogenesis in Pancreatic Cancer by Silencing ROBO1 and SRGAP2.","date":"2022","source":"Journal of immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/36277474","citation_count":24,"is_preprint":false},{"pmid":"23119100","id":"PMC_23119100","title":"Slit2N/Robo1 inhibit HIV-gp120-induced migration and podosome formation in immature dendritic cells by sequestering LSP1 and WASp.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23119100","citation_count":24,"is_preprint":false},{"pmid":"21820427","id":"PMC_21820427","title":"Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance.","date":"2011","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/21820427","citation_count":23,"is_preprint":false},{"pmid":"20071679","id":"PMC_20071679","title":"Effect of Robo1 on retinal pigment epithelial cells and experimental proliferative vitreoretinopathy.","date":"2010","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/20071679","citation_count":22,"is_preprint":false},{"pmid":"27653286","id":"PMC_27653286","title":"Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2.","date":"2016","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/27653286","citation_count":22,"is_preprint":false},{"pmid":"33714986","id":"PMC_33714986","title":"Roundabout homolog 1 inhibits proliferation via the YY1-ROBO1-CCNA2-CDK2 axis in human pancreatic cancer.","date":"2021","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/33714986","citation_count":22,"is_preprint":false},{"pmid":"25326112","id":"PMC_25326112","title":"Inhibition of endothelial Slit2/Robo1 signaling by thalidomide restrains angiogenesis by blocking the PI3K/Akt pathway.","date":"2014","source":"Digestive diseases and sciences","url":"https://pubmed.ncbi.nlm.nih.gov/25326112","citation_count":22,"is_preprint":false},{"pmid":"24247621","id":"PMC_24247621","title":"Frameshift mutations of axon guidance genes ROBO1 and ROBO2 in gastric and colorectal cancers with microsatellite instability.","date":"2013","source":"Pathology","url":"https://pubmed.ncbi.nlm.nih.gov/24247621","citation_count":22,"is_preprint":false},{"pmid":"27893610","id":"PMC_27893610","title":"Slit2/Robo1 promotes synaptogenesis and functional recovery of spinal cord injury.","date":"2017","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/27893610","citation_count":21,"is_preprint":false},{"pmid":"26713366","id":"PMC_26713366","title":"Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/26713366","citation_count":21,"is_preprint":false},{"pmid":"21301049","id":"PMC_21301049","title":"Src activates Abl to augment Robo1 expression in order to promote tumor cell migration.","date":"2010","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/21301049","citation_count":21,"is_preprint":false},{"pmid":"25006547","id":"PMC_25006547","title":"A (90)Y-labelled anti-ROBO1 monoclonal antibody exhibits antitumour activity against hepatocellular carcinoma xenografts during ROBO1-targeted radioimmunotherapy.","date":"2014","source":"EJNMMI research","url":"https://pubmed.ncbi.nlm.nih.gov/25006547","citation_count":21,"is_preprint":false},{"pmid":"29864155","id":"PMC_29864155","title":"Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29864155","citation_count":20,"is_preprint":false},{"pmid":"27176045","id":"PMC_27176045","title":"Slit2 and Robo1 induce opposing effects on metastasis of hepatocellular carcinoma Sk-hep-1 cells.","date":"2016","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/27176045","citation_count":20,"is_preprint":false},{"pmid":"31242633","id":"PMC_31242633","title":"Activation of Slit2/Robo1 Signaling Promotes Tumor Metastasis in Colorectal Carcinoma through Activation of the TGF-β/Smads Pathway.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31242633","citation_count":19,"is_preprint":false},{"pmid":"19084519","id":"PMC_19084519","title":"Robo1/robo4: different expression patterns in retinal development.","date":"2008","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/19084519","citation_count":18,"is_preprint":false},{"pmid":"33621954","id":"PMC_33621954","title":"Raddeanin A inhibits proliferation, invasion, migration and promotes apoptosis of cervical cancer cells via regulating miR-224-3p/Slit2/Robo1 signaling pathway.","date":"2021","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/33621954","citation_count":18,"is_preprint":false},{"pmid":"31448886","id":"PMC_31448886","title":"A Novel Missense Mutation in Human Receptor Roundabout-1 (ROBO1) Gene Associated with Pituitary Stalk Interruption Syndrome.","date":"2019","source":"Journal of clinical research in pediatric endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/31448886","citation_count":17,"is_preprint":false},{"pmid":"28406573","id":"PMC_28406573","title":"Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.","date":"2017","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/28406573","citation_count":17,"is_preprint":false},{"pmid":"26889813","id":"PMC_26889813","title":"MiR-218 inhibits the tumorgenesis and proliferation of glioma cells by targeting Robo1.","date":"2016","source":"Cancer biomarkers : section A of Disease markers","url":"https://pubmed.ncbi.nlm.nih.gov/26889813","citation_count":17,"is_preprint":false},{"pmid":"26362767","id":"PMC_26362767","title":"Slit Binding via the Ig1 Domain Is Essential for Midline Repulsion by Drosophila Robo1 but Dispensable for Receptor Expression, Localization, and Regulation in Vivo.","date":"2015","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/26362767","citation_count":17,"is_preprint":false},{"pmid":"35472309","id":"PMC_35472309","title":"Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division.","date":"2022","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/35472309","citation_count":16,"is_preprint":false},{"pmid":"30288875","id":"PMC_30288875","title":"Implication of SLIT3-ROBO1/ROBO2 in granulosa cell proliferation, differentiation and follicle selection in the prehierarchical follicles of hen ovary.","date":"2018","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/30288875","citation_count":16,"is_preprint":false},{"pmid":"26282852","id":"PMC_26282852","title":"Role of Slit2/Robo1 in trophoblast invasion and vascular remodeling during ectopic tubal pregnancy.","date":"2015","source":"Placenta","url":"https://pubmed.ncbi.nlm.nih.gov/26282852","citation_count":16,"is_preprint":false},{"pmid":"27431199","id":"PMC_27431199","title":"Slit2‑Robo1 signaling promotes the adhesion, invasion and migration of tongue carcinoma cells via upregulating matrix metalloproteinases 2 and 9, and downregulating E‑cadherin.","date":"2016","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/27431199","citation_count":16,"is_preprint":false},{"pmid":"25988316","id":"PMC_25988316","title":"Robo1/2 regulate follicle atresia through manipulating granulosa cell apoptosis in mice.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/25988316","citation_count":16,"is_preprint":false},{"pmid":"35584116","id":"PMC_35584116","title":"An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35584116","citation_count":15,"is_preprint":false},{"pmid":"23438940","id":"PMC_23438940","title":"Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation.","date":"2013","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/23438940","citation_count":15,"is_preprint":false},{"pmid":"29217730","id":"PMC_29217730","title":"In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats.","date":"2018","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/29217730","citation_count":15,"is_preprint":false},{"pmid":"28485101","id":"PMC_28485101","title":"Alterted SLIT2/ROBO1 signalling is linked to impaired placentation of missed and threatened miscarriage in early pregnancy.","date":"2017","source":"Histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/28485101","citation_count":15,"is_preprint":false},{"pmid":"34369900","id":"PMC_34369900","title":"Circ_USP36 Silencing Attenuates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Endothelial Cells in Atherosclerosis Through Mediating miR-197-3p/ROBO1 Axis.","date":"2021","source":"Journal of cardiovascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34369900","citation_count":15,"is_preprint":false},{"pmid":"26610476","id":"PMC_26610476","title":"Epigenetic Repression of miR-218 Promotes Esophageal Carcinogenesis by Targeting ROBO1.","date":"2015","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/26610476","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53627,"output_tokens":11293,"usd":0.165138,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":22014,"output_tokens":9064,"usd":0.168335,"stage2_stop_reason":"end_turn"},"total_usd":0.333473,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"Targeted deletion of exon 5 (eliminating the first Ig domain) of Dutt1/Robo1 in mice causes neonatal respiratory failure due to delayed lung maturation (reduced air spaces, increased mesenchyme) and bronchial epithelial hyperplasia in survivors, establishing a direct functional role for Robo1 in lung development.\",\n      \"method\": \"Targeted gene knockout in mice (homozygous deletion of exon 5); histopathology and phenotypic analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout mouse model with specific developmental phenotype, replicated across homozygous and survivor cohorts in a single rigorous study\",\n      \"pmids\": [\"11734623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Dutt1/Robo1 heterozygous mice spontaneously develop lymphomas and lung adenocarcinomas; tumors retain the structurally normal allele but show promoter methylation and reduced Robo1 protein, consistent with Knudson two-hit tumor suppressor mechanism.\",\n      \"method\": \"Heterozygous knockout mouse model; LOH analysis; bisulfite sequencing of promoter; Western blot and immunohistochemistry for protein expression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (LOH, methylation, protein expression) in a genetic mouse model within a single study\",\n      \"pmids\": [\"15374951\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The DUTT1/ROBO1 promoter CpG island is hypermethylated in breast, lung, and renal cell carcinoma tumors; methylation silences expression, which is reactivated by 5-aza-2'-deoxycytidine treatment, identifying promoter methylation as an epigenetic inactivation mechanism.\",\n      \"method\": \"Bisulfite sequencing; methylation-specific PCR; RT-PCR expression analysis; 5-aza-2'-deoxycytidine demethylation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal epigenetic and expression methods, replicated across multiple cancer types and cell lines\",\n      \"pmids\": [\"12082532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Robo1 knockout mice display major forebrain axon pathfinding defects (corpus callosum and hippocampal commissure dysgenesis, premature corticothalamic/thalamocortical targeting) and up to twice as many cortical interneurons migrate into the cortex, establishing Robo1 as a regulator of both major axon tract formation and interneuron migration.\",\n      \"method\": \"Robo1 knockout mice (targeted deletion of exon 5); MRI diffusion tensor imaging; immunohistochemistry; cell counting\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with multiple distinct phenotypic readouts (axon guidance and cell migration) replicated across developmental time points\",\n      \"pmids\": [\"16690755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RNAi-mediated knockdown of Robo1 in glioma cell lines abolishes the repulsive/migratory-inhibitory effect of recombinant Slit2, demonstrating that Robo1 is the major functional receptor for Slit2-mediated chemorepulsion in glioma cells.\",\n      \"method\": \"siRNA knockdown of Robo1; modified Boyden chamber migration assay with recombinant Slit2\",\n      \"journal\": \"Journal of neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional rescue/loss-of-function assay with defined phenotypic readout, single lab, single method\",\n      \"pmids\": [\"17968499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The C. elegans kinesin-related protein VAB-8L promotes posterior cell/growth-cone migration by increasing surface levels of the Robo1 ortholog SAX-3; the Rac GEF UNC-73 (Trio) physically interacts with both VAB-8L and the intracellular domain of SAX-3, supporting a model in which receptor surface localization is regulated by this complex.\",\n      \"method\": \"C. elegans genetics (epistasis); co-immunoprecipitation; receptor abundance measurement; gain/loss-of-function assays\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic epistasis combined with physical interaction (co-IP) and receptor localization data in a single rigorous study\",\n      \"pmids\": [\"17237778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Robo1 forms a heterodimeric complex with Robo4; Robo1 is required for Robo4-mediated filopodia induction in endothelial cells; the Robo4 intracellular domain binds WASP, N-WASP, and WIP (actin nucleation-promoting factors) as identified by yeast two-hybrid and GST pulldown, linking Robo1/Robo4 signaling to actin-based cell motility.\",\n      \"method\": \"siRNA knockdown; Robo4-GFP overexpression; yeast two-hybrid; GST pulldown; endothelial tube formation and migration assays; co-immunoprecipitation\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, GST pulldown, siRNA functional assay, co-IP) in a single study establishing complex formation and downstream actin effectors\",\n      \"pmids\": [\"18948384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Robo1 and Robo2 have distinct and collaborative roles in post-crossing commissural axon guidance: Robo2 is required for axons to project away from the floor plate into the lateral funiculus, while Robo1 prevents axonal stalling after midline crossing; genetic epistasis with Robo3 mutants further defines their collaborative role in preventing midline re-entry.\",\n      \"method\": \"Genetic combination mutants (Robo1, Robo2, Robo3 single and double/triple knockouts); axon trajectory analysis in mouse spinal cord\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with multiple allele combinations defining distinct roles of each paralog\",\n      \"pmids\": [\"20631173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human ROBO1 undergoes sequential proteolytic cleavage by metalloproteinases (producing ROBO1-CTF1 at 129 kDa) followed by gamma-secretase cleavage (producing ROBO1-CTF2 at 118 kDa); the resulting intracellular fragment accumulates in the nucleus in cancer cells, and nuclear accumulation is abolished by either metalloproteinase inhibitor TAPI-1 or gamma-secretase inhibitor L-685,458.\",\n      \"method\": \"Western blot of cleavage fragments; pharmacological inhibition with TAPI-1 and L-685,458; nuclear fractionation; determination of precise cleavage site\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical identification of cleavage products with pharmacological validation, single lab\",\n      \"pmids\": [\"20471383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SLIT/ROBO1 signaling in the mammary basal cell layer restricts basal cell proliferation by inhibiting canonical WNT signaling, shifting β-catenin from the nuclear pool to cytoplasmic/membrane pools; loss of SLIT/ROBO1 signaling specifically in basal cells results in excess basal cells and precocious branching morphogenesis. TGF-β1 acts upstream of Robo1, inducing its expression in the basal layer.\",\n      \"method\": \"Conditional loss-of-function mouse models; mammary gland whole-mount analysis; immunofluorescence for β-catenin localization; Western blot; TGF-β1 treatment\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional knockout with mechanistic pathway dissection (WNT/β-catenin), multiple orthogonal methods\",\n      \"pmids\": [\"21664580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Slit2-Robo1 signaling inhibits Cdc42 activity in glioma cells, and this is the mechanism by which the pathway suppresses glioma cell invasion; Cdc42-GTP inactivation mediates the anti-invasive effect.\",\n      \"method\": \"Small GTPase pull-down assay (Cdc42 activity); siRNA knockdown; migration/invasion assay\",\n      \"journal\": \"Neuroscience bulletin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single GTPase activity assay with no independent replication; abstract-level detail only\",\n      \"pmids\": [\"21113198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Heparin binds to Robo1 with an affinity of ~650 nM; N-sulfo and 6-O-sulfo groups on heparin are essential for the Robo1-heparin interaction; full-length heparin chains and higher sulfation levels increase binding affinity.\",\n      \"method\": \"Surface plasmon resonance (SPR); chemical modification of heparin; SPR solution competition studies\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro binding assay with systematic chemical modification, single lab\",\n      \"pmids\": [\"23994753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FLRT3 is a novel co-receptor for Robo1; in thalamic axons, Slit1 acting through both Robo1 and FLRT3 induces Netrin-1 attraction by upregulating surface DCC via activation of protein kinase A (PKA); thalamic axons lacking FLRT3 are insensitive to Netrin-1, and loss of FLRT3 causes axon guidance defects in vivo.\",\n      \"method\": \"Co-receptor identification; in vitro axon guidance assays; PKA activation assays; surface DCC measurement; in vivo guidance phenotype analysis in knockout mice\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods establishing a novel co-receptor interaction with defined downstream signaling (PKA/DCC axis)\",\n      \"pmids\": [\"24560577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ROBO1 negatively regulates cell motility and invasiveness of prostate cancer cells through interaction with DOCK1 (via its SH3-SH2 domain), controlling Rac1 activation; loss of ROBO1 leads to Rac1 activation, which destabilizes E-cadherin/β-catenin cytoskeleton and induces cell migration.\",\n      \"method\": \"ROBO1-expressing and ROBO1-C2C3 mutant constructs; co-immunoprecipitation; confocal microscopy; luciferase reporter; migration/invasion assays\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain-mapping mutants and functional assays, single lab\",\n      \"pmids\": [\"24752651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Robo1 knockout in the developing neocortex causes a significant increase in progenitor cells and both early- and late-born pyramidal neuron populations due to prolonged proliferative activity of progenitors; confirmed by in utero electroporation of Robo1 shRNA and in dissociated cortical cell cultures.\",\n      \"method\": \"Robo1 knockout mice; in utero electroporation with shRNA; dissociated cortical cell cultures; BrdU/Ki67 proliferation analysis; layer-specific marker immunostaining\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout replicated by independent shRNA electroporation and in vitro assays within a single study\",\n      \"pmids\": [\"24741061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 acting through Robo1 and Robo2 promotes endothelial cell migration and retinal angiogenesis; Robo1 and Robo2 are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation in retinal endothelial cells.\",\n      \"method\": \"Conditional knockout mice (endothelial-specific deletion of Slit2, Robo1, Robo2); mouse postnatal retinal angiogenesis assay; Rac1 activation assay; lamellipodia measurement; ocular neovascular disease model\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockouts with in vivo and in vitro mechanistic readouts including Rac1 activation and lamellipodia formation\",\n      \"pmids\": [\"25894826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Two distinct heparin-binding sites on Robo1 were identified: one at the previously known site and a second at the disordered N-terminus; mutagenesis of the N-terminal site decreased heparin binding affinity; heparin binding also induces a minor conformational change in the Ig2 domain.\",\n      \"method\": \"Electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting; surface plasmon resonance; heparin affinity chromatography; site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — novel structural footprinting method combined with mutagenesis and SPR quantification in a single rigorous study\",\n      \"pmids\": [\"25752613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SLIT2/ROBO1 signaling regulates mammary stem cell self-renewal by governing the choice between asymmetric and symmetric cell divisions through the transcription factor SNAI1 and spindle orientation protein mINSC; loss of SLIT2/ROBO1 increases nuclear SNAI1, which upregulates mInsc, increasing symmetric divisions and expanding the stem cell pool.\",\n      \"method\": \"Conditional knockout mouse model; single-cell lineage tracing; mInsc/SNAI1 expression analysis; SLIT2 treatment rescue experiments; mammary gland outgrowth assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional genetic model with mechanistic pathway dissection (SNAI1/mInsc axis) and rescue experiments\",\n      \"pmids\": [\"26440891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Activation of Slit2-Robo1 signaling promotes liver fibrosis by activating hepatic stellate cells (HSCs); mechanistically, Robo1 signaling phosphorylates Smad2, Smad3, PI3K, and AKT independently of TGF-β1, and promotes α-SMA expression; Robo1 neutralizing antibody R5 and Robo1 siRNA block these effects in vitro and attenuate CCl4-induced fibrosis in vivo.\",\n      \"method\": \"Slit2 transgenic and Robo1/2 heterozygous knockout mice; CCl4-induced fibrosis model; Robo1 siRNA and neutralizing antibody (R5); Western blot for Smad2/3, PI3K, AKT, α-SMA phosphorylation\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models (transgenic, knockout, siRNA, antibody) with defined signaling pathway readouts in a single study\",\n      \"pmids\": [\"26264936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway; mechanistically, Robo1 activation by Slit2 activates Src, which downregulates E-cadherin, thereby releasing β-catenin to activate Wnt target genes.\",\n      \"method\": \"Slit2 transgenic and ApcMin/+ mouse models; DMH/DSS colorectal carcinoma model; Western blot; immunohistochemistry; pharmacological Src inhibition\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vivo mouse models with defined signaling pathway (Src/E-cadherin/β-catenin), single lab\",\n      \"pmids\": [\"25605242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"srGAP1 is a Robo1-interacting protein that mediates the anti-migratory function of Slit2-Robo1 in colorectal cancer cells by inhibiting Cdc42 activity; co-immunoprecipitation confirmed the Robo1-srGAP1 interaction; immunofluorescence showed similar dynamic subcellular distribution upon Slit2 treatment.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence; Cdc42 GTPase pull-down assay; wound healing migration assay\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating physical interaction with functional GTPase activity assay, single lab\",\n      \"pmids\": [\"27923383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Recombinant Slit2 reduces peripheral immune cell infiltration after surgical brain injury through Robo1-srGAP1-mediated inhibition of Cdc42 activity; decoy Robo1 co-administration and srGAP1 siRNA reversed the protective effects of Slit2, establishing the Robo1-srGAP1-Cdc42 signaling axis in immune cell migration.\",\n      \"method\": \"Rat surgical brain injury model; recombinant Slit2 and decoy Robo1; srGAP1 siRNA; Cdc42 activity assay; brain edema measurement; immunohistochemistry\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo model with decoy receptor and siRNA controls, Cdc42 activity assay; single lab\",\n      \"pmids\": [\"26550694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2 promotes angiogenesis via the Robo1-VEGFR2-ERK1/2 pathway; Slit2 overexpression upregulates Robo1, VEGFR2, and phosphorylated ERK1/2 in both transgenic mice and HUVECs, and promotes endothelial cell proliferation, migration, and tube formation.\",\n      \"method\": \"Slit2 transgenic mice; laser-induced CNV model; HUVEC transfection; Western blot for VEGFR2 and p-ERK1/2; proliferation, migration and tube formation assays\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic mouse model combined with in vitro pathway analysis, single lab\",\n      \"pmids\": [\"26244297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Heparan sulfate octasaccharide ligand requirements for Robo1 were defined: Robo1 preferentially binds structures with 6-O-sulfate esters (critical), whereas 2-O-sulfate esters substantially reduce binding; a high-affinity HS octasaccharide ligand was synthesized and confirmed by SPR to bind Robo1 and reverse Slit2-Robo1-mediated inhibition of endothelial cell migration.\",\n      \"method\": \"HS affinity enrichment; HILIC-MS/MS structural determination; chemical synthesis of HS oligosaccharides; surface plasmon resonance (SPR) binding studies; endothelial cell migration assay\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — integrated chemical synthesis with quantitative SPR binding and functional cell migration assay, rigorous structure-activity relationship study\",\n      \"pmids\": [\"27611601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NMR characterization of Robo1-Ig1-2 interaction with a synthetic heparan sulfate tetramer identified K81 as part of the binding site (Kd ~255 µM); saturation transfer difference and trNOE defined the binding epitope and bound conformation of HS; paramagnetic perturbation localized the binding site; a structural model of the complex was generated.\",\n      \"method\": \"Single amino acid type NMR labeling; STD NMR; trNOE; paramagnetic perturbation; complex modeling\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple NMR methods characterizing binding site, bound conformation, and dissociation constant; single lab\",\n      \"pmids\": [\"27653286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A stiff ECM increases Robo1 levels by downregulating miR-203; SLIT2/ROBO1 signaling stimulates Rac activity and focal adhesion kinase (FAK) to enhance cell tension, maintain cell shape, and facilitate matrix adhesion; Robo1 ablation in the mammary gland compromises actin stress fiber assembly and inhibits cell contractility.\",\n      \"method\": \"miR-203 and Robo1 manipulation; Rac activation assay; FAK activity assay; actin staining; mammary gland conditional knockout; tunable hydrogel substrates\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro with multiple signaling readouts (Rac, FAK, actin); single lab\",\n      \"pmids\": [\"26975850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of Robo1 Ig1-4 and negative-stain EM reconstruction of the full ectodomain show that Robo1 forms compact dimers mediated by central Ig domains, which further interact in a 'back-to-back' fashion to generate tetrameric assemblies; no change in oligomerization is observed upon Slit2-N binding, suggesting Slit2-N induces a conformational change rather than oligomerization change.\",\n      \"method\": \"X-ray crystallography (Robo1 Ig1-4 and Ig5); negative stain electron microscopy; fluorescent imaging with dimeric Slit2-N\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with EM reconstruction and Slit2 binding experiment; single lab but Tier 1 methods\",\n      \"pmids\": [\"29307485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SLIT2/ROBO1 axis promotes the Warburg effect (aerobic glycolysis) in osteosarcoma cells via activation of the SRC/ERK/c-MYC/PFKFB2 signaling pathway; knockdown or overexpression experiments in vitro and in vivo confirmed this metabolic-oncogenic role.\",\n      \"method\": \"siRNA knockdown and overexpression; xenograft mouse model; Western blot for SRC, ERK, c-MYC, PFKFB2; metabolic assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo models with defined signaling pathway; single lab\",\n      \"pmids\": [\"29523788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"USP33 (ubiquitin-specific protease 33) deubiquitinates and stabilizes Robo1; reduced USP33 in gastric cancer leads to increased Robo1 ubiquitination and degradation, diminishing the inhibitory effect of Slit2-Robo1 signaling on cell migration and EMT.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination protein analysis; siRNA knockdown; in vitro and in vivo migration/EMT assays\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating physical interaction and ubiquitination assay establishing USP33 as a deubiquitinase for Robo1; single lab\",\n      \"pmids\": [\"30896071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"miR-92 suppresses Robo1 translation (without mRNA degradation) in commissural neurons by binding the Robo1 3'UTR; disrupting miR-92/Robo1 interaction induces premature Slit2 repulsion and causes commissural axon projection defects in vivo; this mechanism regulates the temporal control of Slit sensitivity during midline crossing.\",\n      \"method\": \"miR-92 overexpression and inhibition; luciferase 3'UTR reporter assay; in vitro Slit2 repulsion assay; in vivo commissural axon labeling in chick embryo\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — 3'UTR reporter validation combined with in vitro repulsion assay and in vivo axon guidance phenotype in a single study\",\n      \"pmids\": [\"30184503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Alternative splicing at a conserved microexon in mammalian ROBO1 and ROBO2, regulated by NOVA splicing factors, generates isoforms with distinct guidance activities; precrossing ROBO1 isoforms are inhibited to allow midline crossing, while postcrossing disinhibition prevents midline re-entry; this provides temporal regulation of Robo-mediated axonal repulsion independent of protein expression levels.\",\n      \"method\": \"Alternative splicing analysis; NOVA factor manipulation; in vitro Slit repulsion assay; commissural axon trajectory analysis in vivo\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combination of splicing factor manipulation, isoform-specific functional assays, and in vivo guidance phenotype analysis\",\n      \"pmids\": [\"31392959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Slit2/Robo1 signaling promotes tumor metastasis in colorectal carcinoma partly through activation of the TGF-β/Smads pathway; blocking Slit2-Robo1 binding inactivates TGF-β/Smads and inhibits tumor cell migration, which can be partially restored by exogenous TGF-β1.\",\n      \"method\": \"Robo1 blocking antibody; TGF-β1 rescue experiment; Smad phosphorylation Western blot; migration assay; in vivo tumor models\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor blockade with TGF-β1 rescue experiment establishing pathway ordering; single lab\",\n      \"pmids\": [\"31242633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SLIT2 inhibits macropinocytosis in macrophages and RAS-transformed cancer cells through Robo1; mechanistically, SLIT2/ROBO1 signaling induces cytoskeletal changes that oppose cortical cytoskeletal remodeling required for macropinocytosis; in vivo, SLIT2 attenuates muramyl dipeptide uptake, preventing NOD2-dependent NF-κB activation and CXCL1 secretion.\",\n      \"method\": \"In vitro macropinocytosis assays; in vivo mouse model with SLIT2 treatment; blocking endogenous SLIT2; cytoskeletal imaging; NOD2/NF-κB pathway analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro and in vivo experiments with multiple mechanistic readouts (cytoskeletal, NF-κB, CXCL1); rigorous study\",\n      \"pmids\": [\"32807784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PRRG4 promotes breast cancer metastasis by recruiting E3 ubiquitin ligase NEDD4 (through LPSY and PPPY motifs) to ubiquitinate and degrade Robo1, thereby activating Src and FAK signaling and promoting invasion.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; PRRG4 knockdown/overexpression; experimental metastasis model; Western blot for Src/FAK phosphorylation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with domain mapping, ubiquitination assay establishing NEDD4 as E3 ligase for Robo1; single lab\",\n      \"pmids\": [\"33037408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Src kinase activates Abl kinase, which stabilizes Robo1 protein to promote cell migration; inhibition of Abl by siRNA or kinase inhibitors decreases Robo1 protein levels and suppresses migration of transformed cells; Robo1 utilizes Cdc42 and Rac1 GTPases downstream to induce cell migration.\",\n      \"method\": \"Abl siRNA knockdown; pharmacological kinase inhibitors; Western blot for Robo1 protein levels; migration assay; small GTPase activation assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and pharmacological inhibition with defined mechanistic readouts (Robo1 stability, GTPase activity); single lab\",\n      \"pmids\": [\"21301049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Slit2N/Robo1 inhibits HIV-gp120-induced migration and podosome formation in immature dendritic cells by causing Robo1 to sequester WASp and LSP1 away from β-actin, preventing WASp-Arp2/3-β-actin complex formation; Slit2N also inhibits Src signaling and downstream Rac1, Pyk2, paxillin, and CDC42 activation.\",\n      \"method\": \"Co-immunoprecipitation; transendothelial migration assay; podosome formation assay; Rac1/CDC42 activation assay; Src signaling Western blot\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating Robo1-WASp/LSP1 sequestration with functional migration and podosome readouts; single lab\",\n      \"pmids\": [\"23119100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2/Robo1 signaling is required for Drosophila Robo1 midline repulsion; Ig1 domain deletion disrupts Slit binding in vitro and abolishes ectopic midline repulsion in gain-of-function studies; Ig1 is not required for proper Robo1 expression, axonal localization, or Commissureless-dependent regulation in vivo.\",\n      \"method\": \"In vitro Slit binding assay in Drosophila cells; Drosophila in vivo gain-of-function and genetic rescue assays; immunostaining for axonal localization and Comm-dependent regulation\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro binding assay combined with in vivo genetic rescue, domain deletion analysis establishing Ig1 as essential for Slit-dependent activity\",\n      \"pmids\": [\"26362767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Fn3 domain deletion in Drosophila Robo1 impairs exclusion of Robo1 from commissures and reduces Commissureless-dependent downregulation of Robo1, while Fn1 and Fn2 deletions do not affect Slit binding, expression, midline repulsive function, or axonal localization; all three Fn repeats are individually dispensable for midline repulsion itself.\",\n      \"method\": \"In vivo domain deletion analysis (Fn1, Fn2, Fn3); in vitro Slit binding assay; midline repulsion genetic rescue assay; immunostaining for localization and Comm-dependent regulation\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion series with in vitro and in vivo functional assays, single lab\",\n      \"pmids\": [\"29217730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Robo1 promotes angiogenesis in hepatocellular carcinoma by promoting Cdc42 expression in endothelial cells; knockdown of Robo1 distorts the actin cytoskeleton in HUVECs; in vivo, overexpression of Robo1 in endothelial cells mixed with hepatoma cells enhances tumor growth and microvessel density.\",\n      \"method\": \"Robo1 overexpression and knockdown in HUVECs; nude mouse tumor implantation; Cdc42 expression analysis; actin cytoskeleton imaging\",\n      \"journal\": \"Tumour biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo experiments with Cdc42 mechanistic readout; single lab\",\n      \"pmids\": [\"26022159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Slit2/Robo1 signaling inhibits HUVEC migration in a Robo4-dependent manner; Robo4 negatively regulates the Slit2-Robo1 migratory signal through an intracellular mechanism; CdGAP (a Rac1/Cdc42-specific GTPase activating protein) was identified by targeted proteomics as a Robo1-interacting protein and co-immunoprecipitated with Robo1.\",\n      \"method\": \"Boyden chamber migration assay; Robo1 and Robo4 siRNA knockdown; Robo1 extracellular domain blocking; targeted proteomics; co-immunoprecipitation of Robo1-CdGAP\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP identifying CdGAP as Robo1 partner combined with functional migration assay; single lab\",\n      \"pmids\": [\"26713366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"YY1 transcription factor directly binds the ROBO1 promoter to promote transcription, as demonstrated by luciferase reporter, ChIP, and EMSA assays; ROBO1 inhibits pancreatic cancer cell proliferation through the CCNA2/CDK2 axis, causing S-phase arrest.\",\n      \"method\": \"Luciferase reporter gene assay; chromatin immunoprecipitation (ChIP); electrophoretic mobility shift assay (EMSA); cell cycle analysis; subcutaneous xenograft in nude mice\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — three orthogonal assays (luciferase, ChIP, EMSA) confirming YY1-ROBO1 promoter interaction, plus functional downstream pathway (CCNA2/CDK2); single lab but multiple rigorous methods\",\n      \"pmids\": [\"33714986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Glial-secreted Netrins regulate Drosophila Robo1 signaling threshold levels in neural stem cells; Netrin-Frazzled/DCC signaling modulates Robo1 levels through Abelson kinase; unbalanced Robo1 signaling impairs asymmetric stem cell division by dysregulating aPKC, Canoe/Afadin, and Numb through Rac1 and Cdc42.\",\n      \"method\": \"Drosophila genetics (Netrin, Frazzled/DCC, Abelson kinase, Robo1 manipulations); immunostaining for ASCD machinery components; larval brain neuroblast lineage analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in Drosophila model with defined downstream effectors (Rac1/Cdc42/aPKC axis); single lab\",\n      \"pmids\": [\"35472309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ROBO1 exhibits dependence receptor (DR) characteristics in pancreatic ductal adenocarcinoma liver metastasis; PTP4A2 (protein tyrosine phosphatase 4A2) dephosphorylates ROBO1 in recurrent glioblastoma to modulate its downstream signaling and tumorigenicity; genetic or pharmacological inhibition of PTP4A2 acts through ROBO1; ROBO1-targeted CAR T cells eradicate tumors in xenograft models.\",\n      \"method\": \"CRISPR genetic perturbation screens; PTP4A2 inhibition; ROBO1 dephosphorylation assay; CDX xenograft models; CAR T cell therapy\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screens combined with mechanistic validation (dephosphorylation axis, in vivo CAR T efficacy); single study\",\n      \"pmids\": [\"39095594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SLIT2-ROBO1-mediated coadaptation facilitates implantation and outgrowth of pancreatic ductal adenocarcinoma disseminated tumor cells in the liver premetastatic niche; ROBO1 displays dependence receptor characteristics; a ROBO1-neutralizing antibody significantly attenuates liver metastasis.\",\n      \"method\": \"Mouse premetastatic/macrometastatic niche models; CT/3D bioluminescence imaging; ROBO1 neutralizing antibody; human PDAC patient samples; mechanistic dependence receptor characterization\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo mouse models with antibody intervention and defined mechanistic characteristic (dependence receptor); single lab\",\n      \"pmids\": [\"36792623\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ROBO1 is a transmembrane immunoglobulin superfamily receptor that binds Slit ligands (via its Ig1 domain, facilitated by heparan sulfate co-receptors binding at two distinct sites) and signals through intracellular interactions with effectors including srGAP1, DOCK1, CdGAP, and WASP-family proteins to regulate Cdc42/Rac1 GTPase activity, actin cytoskeletal remodeling, and cell migration; it forms homo- and hetero-dimeric (with Robo4) ectodomain assemblies and undergoes metalloproteinase/gamma-secretase sequential cleavage with nuclear translocation of its intracellular fragment; its expression is regulated by promoter methylation, ubiquitination via NEDD4 (counteracted by USP33 deubiquitylation), Abl kinase-mediated stabilization, post-transcriptional repression by multiple miRNAs (miR-218, miR-92, miR-203, miR-219), YY1 transcriptional activation, and alternative splicing of a conserved microexon; in vivo, Robo1 is essential for midline axon guidance, cortical interneuron migration, forebrain commissure formation, neocortical progenitor proliferation, lung development, and mammary branching morphogenesis, and its downstream pathways include WNT/β-catenin, TGF-β/Smad, PI3K/AKT, Src/ERK, and PKA/DCC signaling axes depending on cellular context.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROBO1 is a transmembrane immunoglobulin-superfamily receptor for secreted Slit ligands that governs cell migration, axon guidance, and tissue morphogenesis by transducing repulsive cues into actin cytoskeletal reorganization [#3, #36]. Ligand engagement requires the first Ig domain, which is essential for Slit binding and for repulsive activity in vivo [#36], and is potentiated by heparan/heparin co-receptors that bind Robo1 at two distinct sites — a previously known site and a second within the disordered N-terminus — with 6-O-sulfation being critical for high-affinity recognition [#11, #16, #23]. Structurally, the Robo1 ectodomain assembles into compact dimers and back-to-back tetramers via central Ig domains, with Slit2-N binding inducing a conformational change rather than altering oligomeric state, and Robo1 also forms heterodimers with Robo4 [#26, #6]. Intracellularly, Robo1 controls the Cdc42/Rac1 GTPase switch through partners including srGAP1, DOCK1, CdGAP, and WASP-family proteins to direct actin remodeling, lamellipodia/filopodia formation, focal-adhesion and cell-tension signaling [#20, #13, #39, #6, #25]. In neural development Robo1 prevents post-crossing commissural axon stalling and midline re-entry, restrains cortical interneuron migration, and limits neocortical progenitor proliferation, with its repulsive output temporally tuned by miR-92 translational repression and NOVA-regulated microexon splicing rather than by expression level alone [#3, #7, #14, #29, #30]. Beyond the nervous system Robo1 directs lung maturation, mammary stem-cell self-renewal and branching via WNT/\\u03b2-catenin and SNAI1/mINSC control, retinal and tumor angiogenesis through Rac1 and VEGFR2/ERK, and immune-cell migration and macropinocytosis [#0, #9, #17, #15, #32]. In cancer Robo1 behaves as a context-dependent tumor suppressor silenced by promoter hypermethylation and destabilized by NEDD4-mediated ubiquitination opposed by USP33 deubiquitylation, and its protein level and signaling are further set by Abl-mediated stabilization, PTP4A2 dephosphorylation, and YY1 transcriptional activation [#1, #2, #33, #28, #34, #42, #40]. Human ROBO1 also undergoes sequential metalloproteinase and gamma-secretase cleavage producing an intracellular fragment that accumulates in the nucleus of cancer cells [#8].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that Robo1 has an essential developmental function beyond neural tissue, demonstrating it is required for lung maturation and epithelial homeostasis.\",\n      \"evidence\": \"Targeted exon-5 (Ig1) knockout in mice with histopathology\",\n      \"pmids\": [\"11734623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the Slit ligand or signaling effectors in lung\", \"Mechanism linking receptor loss to delayed alveolar maturation unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified an epigenetic inactivation mechanism, showing ROBO1 is silenced by promoter CpG-island hypermethylation in multiple carcinomas and reactivated by demethylation.\",\n      \"evidence\": \"Bisulfite sequencing, MSP, and 5-aza-dC reactivation in tumor lines\",\n      \"pmids\": [\"12082532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish downstream tumor-suppressive signaling\", \"Causal contribution to tumor initiation not shown in this study\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Provided in vivo genetic evidence that Robo1 acts as a two-hit tumor suppressor, with the retained allele silenced by methylation in spontaneous tumors.\",\n      \"evidence\": \"Heterozygous knockout mice with LOH, methylation, and protein analyses\",\n      \"pmids\": [\"15374951\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tumor-suppressive effector pathway not defined\", \"Tissue-specificity of suppression unexplained\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined Robo1's dual neural role, showing it both shapes forebrain commissural axon tracts and restrains cortical interneuron migration.\",\n      \"evidence\": \"Robo1 knockout mice with DTI-MRI, immunohistochemistry, cell counting\",\n      \"pmids\": [\"16690755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate cell-autonomous from non-autonomous effects\", \"Intracellular signaling not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected receptor surface availability to migration outcome, showing a kinesin/Trio-GEF complex controls SAX-3/Robo1 surface levels and physically engages the intracellular domain.\",\n      \"evidence\": \"C. elegans epistasis, co-IP, and receptor abundance measurement\",\n      \"pmids\": [\"17237778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian relevance of VAB-8L/UNC-73 mechanism not tested here\", \"Direct effect on GTPase output not measured\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated that Robo1 is the principal functional receptor mediating Slit2 chemorepulsion in tumor cells.\",\n      \"evidence\": \"siRNA knockdown with Boyden-chamber migration in glioma lines\",\n      \"pmids\": [\"17968499\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method, single cell type\", \"Downstream effectors not identified in this study\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked Robo1 to actin-nucleation machinery, showing it heterodimerizes with Robo4 and that the receptor pair connects to WASP/N-WASP/WIP for endothelial filopodia.\",\n      \"evidence\": \"Y2H, GST pulldown, siRNA, co-IP, endothelial assays\",\n      \"pmids\": [\"18948384\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"WASP-family binding mapped to Robo4 ICD, not Robo1 directly\", \"In vivo requirement not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved how the Robo paralogs divide labor at the midline, with Robo1 specifically preventing post-crossing axon stalling and midline re-entry.\",\n      \"evidence\": \"Robo1/2/3 combinatorial knockout mice, spinal axon tracing\",\n      \"pmids\": [\"20631173\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of paralog-specific output not defined\", \"Ligand-specificity at the midline not dissected here\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed human ROBO1 is processed by regulated intramembrane proteolysis, generating a nuclear-accumulating intracellular fragment in cancer cells.\",\n      \"evidence\": \"Cleavage-fragment Western blots, TAPI-1/L-685,458 inhibition, nuclear fractionation\",\n      \"pmids\": [\"20471383\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear function of the ICD fragment not determined\", \"Single-lab biochemistry without in vivo confirmation\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established post-translational control of Robo1 abundance, with Src-activated Abl kinase stabilizing the protein to drive migration via Cdc42/Rac1.\",\n      \"evidence\": \"Abl siRNA/inhibitors, Robo1 Western blots, GTPase activation assays\",\n      \"pmids\": [\"21301049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation site not mapped\", \"Single-lab pharmacological evidence\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed Robo1 in a TGF-\\u03b2-to-WNT regulatory axis controlling tissue branching, showing it restricts mammary basal-cell proliferation by limiting nuclear \\u03b2-catenin.\",\n      \"evidence\": \"Conditional knockout mice, whole-mount and \\u03b2-catenin immunofluorescence, TGF-\\u03b21 treatment\",\n      \"pmids\": [\"21664580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link from receptor to \\u03b2-catenin partitioning not fully resolved\", \"Direct effectors downstream of Robo1 not identified here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined a sequestration mechanism, showing Slit2/Robo1 captures WASp and LSP1 away from \\u03b2-actin to block podosome formation and migration.\",\n      \"evidence\": \"Co-IP, transendothelial migration, podosome and Rac1/CDC42 assays in dendritic cells\",\n      \"pmids\": [\"23119100\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect Robo1-WASp binding not distinguished\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Began mapping the heparin-binding biochemistry of Robo1, quantifying affinity and the sulfation groups required for co-receptor engagement.\",\n      \"evidence\": \"SPR with systematically modified heparin\",\n      \"pmids\": [\"23994753\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding-site location not yet mapped\", \"Functional consequence for Slit signaling not tested here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided anti-invasive mechanism in tumor cells, linking Slit2-Robo1 to suppression of Cdc42 activity.\",\n      \"evidence\": \"Cdc42 pull-down, siRNA, invasion assay in glioma\",\n      \"pmids\": [\"21113198\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single GTPase assay without independent replication\", \"Abstract-level mechanistic detail only\", \"Direct GAP/GEF effector not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified DOCK1 as a Robo1 intracellular effector controlling Rac1 and cadherin-based adhesion, explaining anti-migratory tumor-suppressor activity.\",\n      \"evidence\": \"Domain-mapping mutants, co-IP, confocal, invasion assays in prostate cancer\",\n      \"pmids\": [\"24752651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance not tested\", \"Single-lab co-IP without reciprocal validation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed cross-talk between guidance systems, showing FLRT3 acts as a Robo1 co-receptor allowing Slit1 to switch on Netrin attraction via PKA/DCC.\",\n      \"evidence\": \"Co-receptor identification, axon guidance and PKA/surface-DCC assays, knockout mice\",\n      \"pmids\": [\"24560577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Robo1-FLRT3 association not resolved\", \"Generality beyond thalamic axons unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established a proliferative-control role in cortical development, showing Robo1 loss prolongs progenitor proliferation and expands neuron output.\",\n      \"evidence\": \"Knockout mice, in utero shRNA electroporation, BrdU/Ki67 analysis\",\n      \"pmids\": [\"24741061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling pathway coupling Robo1 to progenitor cell cycle not defined\", \"Ligand source in vivo not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated a pro-angiogenic role, showing endothelial Robo1/Robo2 are required for Slit2- and VEGF-induced Rac1 activation and retinal vessel growth.\",\n      \"evidence\": \"Endothelial conditional knockouts, retinal angiogenesis and Rac1/lamellipodia assays\",\n      \"pmids\": [\"25894826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of VEGF/Robo cross-talk not fully resolved\", \"Direct Rac GEF not identified here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified srGAP1 as the Robo1 effector mediating anti-migratory Cdc42 inhibition in cancer and immune-cell contexts.\",\n      \"evidence\": \"Co-IP, immunofluorescence, Cdc42 pull-down, migration and brain-injury models\",\n      \"pmids\": [\"27923383\", \"26550694\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal validation of the interaction limited\", \"Single-lab co-IP evidence\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed Robo1 governs stem-cell division mode, controlling asymmetric vs symmetric divisions through SNAI1/mINSC and spindle orientation.\",\n      \"evidence\": \"Conditional knockout, single-cell lineage tracing, SLIT2 rescue\",\n      \"pmids\": [\"26440891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link from receptor to SNAI1 not defined\", \"Generality to other stem-cell systems untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Documented context-dependent pro-fibrotic and pro-oncogenic signaling, with Robo1 activating Smad2/3, PI3K/AKT, and Src/Wnt/ERK axes.\",\n      \"evidence\": \"Transgenic/knockout mice, neutralizing antibody, pathway Western blots in liver, intestine, and angiogenesis models\",\n      \"pmids\": [\"26264936\", \"25605242\", \"26244297\", \"26022159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apparent opposite (tumor-promoting vs suppressing) roles not reconciled mechanistically\", \"Direct vs indirect pathway activation unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Expanded the Robo1 effector network, identifying CdGAP as a partner and Robo4 as a negative modulator of the Slit2-Robo1 migratory signal.\",\n      \"evidence\": \"Targeted proteomics, co-IP, Robo1/Robo4 siRNA migration assays\",\n      \"pmids\": [\"26713366\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CdGAP interaction not reciprocally validated\", \"In vivo relevance untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Validated the Ig1 domain as the obligatory Slit-binding and repulsion module while showing it is dispensable for receptor localization and Comm regulation.\",\n      \"evidence\": \"Drosophila in vitro Slit binding and in vivo gain-of-function/rescue with Ig1 deletion\",\n      \"pmids\": [\"26362767\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address co-receptor contributions to binding\", \"Mammalian Ig1 in vivo requirement inferred, not shown here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the structural basis of Robo1-heparan sulfate recognition, defining two binding sites, the critical 6-O-sulfate requirement, and a synthetic ligand that reverses Slit2-Robo1 migration inhibition.\",\n      \"evidence\": \"Hydroxyl-radical footprinting, NMR (STD/trNOE), SPR, HS synthesis, mutagenesis, migration assay\",\n      \"pmids\": [\"25752613\", \"27611601\", \"27653286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of N-terminal HS site in vivo not established\", \"How HS binding alters Slit-dependent signaling kinetics unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Tied mechanical ECM cues to Robo1 expression and cytoskeletal tension, showing matrix stiffness raises Robo1 via miR-203 to drive Rac/FAK-dependent contractility.\",\n      \"evidence\": \"miR-203/Robo1 manipulation, Rac/FAK assays, conditional knockout, tunable hydrogels\",\n      \"pmids\": [\"26975850\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct miR-203/Robo1 targeting versus indirect regulation not fully separated\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the ectodomain architecture, showing Robo1 forms dimers and back-to-back tetramers and that Slit2-N triggers conformation rather than oligomerization change.\",\n      \"evidence\": \"X-ray crystallography of Ig1-4/Ig5 and negative-stain EM with Slit2-N imaging\",\n      \"pmids\": [\"29307485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the conformational change for signaling not shown\", \"Full-length receptor in membrane not visualized\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified Robo1 ubiquitination control, showing USP33 deubiquitinates and stabilizes Robo1, with its loss reducing Slit2-Robo1 tumor-suppressive signaling.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, siRNA, migration/EMT models in gastric cancer\",\n      \"pmids\": [\"30896071\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites not mapped\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked Robo1 to tumor metabolism, showing SLIT2/ROBO1 drives the Warburg effect via SRC/ERK/c-MYC/PFKFB2.\",\n      \"evidence\": \"Knockdown/overexpression, xenografts, pathway Western blots, metabolic assays\",\n      \"pmids\": [\"29523788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct receptor-to-SRC coupling not demonstrated\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established translational, temporal control of Robo1 in axon guidance, showing miR-92 represses Robo1 translation to delay Slit2 sensitivity until after midline crossing.\",\n      \"evidence\": \"miR-92 gain/loss, 3'UTR luciferase reporter, in vitro repulsion, in vivo chick axon labeling\",\n      \"pmids\": [\"30184503\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream control of miR-92 timing not defined\", \"Mammalian conservation of timing not tested here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a second layer of temporal regulation, showing NOVA-controlled microexon splicing generates Robo1 isoforms with distinct repulsive activities independent of expression level.\",\n      \"evidence\": \"Splicing analysis, NOVA manipulation, isoform repulsion assays, in vivo axon trajectories\",\n      \"pmids\": [\"31392959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of isoform activity difference unresolved\", \"Regulation of NOVA timing in vivo not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed a pro-metastatic signaling output in carcinoma, with Slit2-Robo1 activating TGF-\\u03b2/Smad to drive migration.\",\n      \"evidence\": \"Robo1 blocking antibody, TGF-\\u03b21 rescue, Smad phosphorylation, in vivo tumor models\",\n      \"pmids\": [\"31242633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct receptor-to-Smad coupling not shown\", \"Reconciliation with tumor-suppressor role absent\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended Robo1 function to innate immunity, showing SLIT2/ROBO1 blocks macropinocytosis and downstream NOD2/NF-\\u03baB inflammatory signaling by opposing cortical cytoskeletal remodeling.\",\n      \"evidence\": \"Macropinocytosis assays, cytoskeletal imaging, in vivo SLIT2 treatment, NF-\\u03baB/CXCL1 analysis\",\n      \"pmids\": [\"32807784\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoskeletal effectors linking Robo1 to macropinosome closure not fully identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified an E3-ligase axis controlling Robo1 levels, with PRRG4 recruiting NEDD4 to ubiquitinate and degrade Robo1, de-repressing Src/FAK and invasion.\",\n      \"evidence\": \"Co-IP with motif mapping, ubiquitination assay, metastasis model, Src/FAK Western blots\",\n      \"pmids\": [\"33037408\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NEDD4 ubiquitination sites on Robo1 not mapped\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined transcriptional activation and a cell-cycle effector arm, showing YY1 directly drives ROBO1 transcription and ROBO1 arrests pancreatic cancer cells via the CCNA2/CDK2 axis.\",\n      \"evidence\": \"Luciferase, ChIP, EMSA, cell-cycle analysis, xenografts\",\n      \"pmids\": [\"33714986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link from receptor signaling to CCNA2/CDK2 regulation not mechanistically traced\", \"Generality beyond pancreatic cancer untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed Robo1 signaling threshold sets stem-cell division asymmetry, with glial Netrin-Frazzled/DCC tuning Robo1 via Abelson kinase to control aPKC/Canoe/Numb through Rac1/Cdc42.\",\n      \"evidence\": \"Drosophila genetics and ASCD-component immunostaining in neuroblasts\",\n      \"pmids\": [\"35472309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian conservation untested\", \"Direct molecular link from Robo1 level to polarity machinery unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Characterized Robo1 as a dependence receptor and actionable target in pancreatic and brain cancer, with PTP4A2 dephosphorylating ROBO1 and ROBO1-directed antibodies/CAR-T attenuating metastasis and tumor growth.\",\n      \"evidence\": \"CRISPR screens, PTP4A2 inhibition, dephosphorylation assays, premetastatic-niche models, neutralizing antibody and CAR-T xenografts\",\n      \"pmids\": [\"39095594\", \"36792623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dependence-receptor death signaling mechanism not fully defined\", \"PTP4A2 dephosphorylation sites on ROBO1 not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single Slit-Robo1 receptor produces context-opposite outcomes — tumor suppression versus promotion, repulsion versus attraction, proliferation restraint versus angiogenesis — through differential effector engagement, proteolysis, and isoform usage remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking effector choice to opposite cellular outputs\", \"Nuclear function of the cleaved ICD fragment unknown\", \"In vivo significance of ectodomain tetramerization and dual HS sites untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [36, 4, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [20, 13, 39, 34]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [6, 25, 35]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [11, 16, 23, 24]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 5, 26]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [6, 25, 38]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 3, 7, 14, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 18, 19, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 2, 42, 43]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [32, 35, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SLIT2\", \"ROBO4\", \"srGAP1\", \"DOCK1\", \"CdGAP\", \"USP33\", \"NEDD4\", \"FLRT3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}