{"gene":"ARHGAP4","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2002,"finding":"Recombinant ARHGAP4 stimulates the GTPase activity of Rac1, Cdc42, and RhoA in vitro, establishing it as a functional GAP for multiple Rho family members.","method":"In vitro GTPase activity assay with recombinant protein","journal":"Brain research. Molecular brain research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic assay with recombinant protein on three substrates, single lab","pmids":["12414125"],"is_preprint":false},{"year":2002,"finding":"Endogenous ARHGAP4 localizes to the Golgi complex and can redistribute to microtubules during mitosis; it is also detected at the tips of differentiating neurites in PC12 cells.","method":"Subcellular fractionation and immunofluorescence/localization studies","journal":"Brain research. Molecular brain research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization by direct imaging of endogenous protein, single lab, two cell contexts","pmids":["12414125"],"is_preprint":false},{"year":2007,"finding":"The FCH domain of ARHGAP4 is required for localizing the protein to the leading edges of migrating NIH/3T3 cells and to axon growth cones, while the GAP domain and C-terminus are necessary for ARHGAP4-mediated inhibition of cell migration and axon outgrowth.","method":"Structure/function analysis with domain deletion constructs; live-cell migration assay and hippocampal axon outgrowth assay","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis combined with functional migration and axon outgrowth assays, single lab","pmids":["17804252"],"is_preprint":false},{"year":2007,"finding":"Overexpression of ARHGAP4 inhibits NIH/3T3 cell migration and hippocampal axon outgrowth, establishing a functional role as an inhibitor of cell and axon motility.","method":"Overexpression in NIH/3T3 cells (migration assay) and hippocampal neurons (axon outgrowth assay)","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined loss/gain-of-function with specific phenotypic readouts, single lab","pmids":["17804252"],"is_preprint":false},{"year":2019,"finding":"ARHGAP4 interacts with and promotes ubiquitination of HDAC2, which in turn inhibits β-catenin activation, suppressing MMP2/MMP9 expression and pancreatic cancer cell invasion and migration.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, pharmacological inhibitors (CAY10683, XAV939), invasion/migration assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional rescue with inhibitors, single lab, multiple orthogonal methods","pmids":["30958531"],"is_preprint":false},{"year":2019,"finding":"ARHGAP4 overexpression inhibits cell viability, glucose uptake, lactate release, PKM2 expression, and mTOR/HIF-1α pathway activation in pancreatic cancer cells; mTOR inhibitor (rapamycin) or HIF-1α inhibitor (YC-1) rescues the morphological changes induced by ARHGAP4 downregulation, placing ARHGAP4 upstream of the mTOR/HIF-1α axis in the Warburg effect.","method":"Lentiviral overexpression and siRNA knockdown; pharmacological inhibition of mTOR and HIF-1α; glucose uptake and lactate release assays; Western blotting","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via pharmacological rescue combined with gain/loss-of-function, single lab","pmids":["31303760"],"is_preprint":false},{"year":2020,"finding":"miR-939-5p directly targets and suppresses ARHGAP4 expression (validated by luciferase reporter assay), thereby promoting pancreatic cancer cell viability, invasion, and migration; ARHGAP4 overexpression reverses these effects.","method":"Luciferase reporter assay, RT-PCR, Western blot, Transwell invasion/migration assay, rescue experiment","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct target validation by luciferase reporter plus functional rescue, single lab","pmids":["32021284"],"is_preprint":false},{"year":2020,"finding":"ARHGAP4 suppresses epithelial-to-mesenchymal transition (EMT) in human mammary epithelial cells, regulating epithelial/mesenchymal marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven force generation; Septin9 was identified by proteomics as a negative regulator of ARHGAP4 that promotes EMT via FAK/Src signaling.","method":"Comprehensive RhoGAP siRNA screen, proteomic analysis, cell morphology/migration assays, traction force microscopy, 3D morphogenesis assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multidisciplinary functional assays plus proteomic identification of binding partner, single lab","pmids":["32378260"],"is_preprint":false},{"year":2021,"finding":"ARHGAP4 forms a complex with SEPT2 and SEPT9 via its RhoGAP domain and SH3 domain; silencing ARHGAP4 or overexpressing SEPT2/SEPT9 independently induces reorganization of focal adhesions with upregulation of Integrin Beta 1, enhancing cell migration and invasion in a microenvironment-dependent manner.","method":"Co-immunoprecipitation, domain mapping, siRNA knockdown, overexpression, focal adhesion analysis, migration/invasion assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with domain mapping plus multiple orthogonal functional assays, replicated across perturbations (KD and OE of multiple complex members)","pmids":["34524873"],"is_preprint":false},{"year":2023,"finding":"ARHGAP4 binds p53 to inhibit DRAM1 expression in AML cells; ARHGAP4 knockdown activates DRAM1 signaling and induces apoptosis, while DRAM1 knockdown rescues the defects caused by ARHGAP4 deletion, placing ARHGAP4 upstream of p53/DRAM1 in AML leukemogenesis.","method":"Co-immunoprecipitation (ARHGAP4-p53 interaction), siRNA knockdown, in vivo AML progression model, apoptosis assay, colony formation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus genetic epistasis (DRAM1 KD rescue), in vivo validation, single lab","pmids":["37443303"],"is_preprint":false},{"year":2024,"finding":"ARHGAP4 knockdown inhibits migration and invasion of colon cancer cells and decreases expression of TGF-β1, p-Smad2, and p-Smad3, while increasing E-cadherin and decreasing N-cadherin/Vimentin, placing ARHGAP4 upstream of the TGF-β/Smad pathway and EMT in colon cancer.","method":"siRNA knockdown, Western blotting for TGF-β/Smad pathway proteins and EMT markers, Transwell migration/invasion assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single set of methods, no direct binding or rescue experiments","pmids":["38805788"],"is_preprint":false},{"year":2024,"finding":"ARHGAP4 promotes apoptosis and inflammation in ovarian granulosa cells via the PI3K-Akt signaling pathway.","method":"CCK-8 assay, annexin-V/PI staining, ELISA, Western blot for PI3K-Akt pathway components, cell line and clinical tissue samples","journal":"Journal of ovarian research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited mechanistic depth, no direct binding or epistasis experiments","pmids":["38355537"],"is_preprint":false},{"year":2025,"finding":"ARHGAP4 drives colorectal cancer stemness through a positive feedback loop with MYH9/β-catenin/c-Jun, as demonstrated by co-immunoprecipitation (ARHGAP4-MYH9 interaction), chromatin immunoprecipitation (c-Jun binding), and FRAP.","method":"Co-immunoprecipitation, chromatin immunoprecipitation, FRAP, in vitro and in vivo stemness assays, bioinformatics","journal":"NPJ precision oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, ChIP, FRAP) in single lab study","pmids":["40817404"],"is_preprint":false}],"current_model":"ARHGAP4 is a multi-domain RhoGAP (FCH–GAP–SH3) that stimulates GTPase activity of Rac1, Cdc42, and RhoA; its FCH domain targets it to leading edges and growth cones where it inhibits cell migration and axon outgrowth, while its GAP and SH3 domains mediate assembly of an ARHGAP4–SEPT2–SEPT9 complex that bidirectionally modulates integrin-β1-dependent focal adhesions; in addition to its canonical GAP function, ARHGAP4 acts as a scaffolding/regulatory protein that ubiquitinates HDAC2 to suppress β-catenin/Wnt signaling, binds p53 to repress DRAM1-dependent apoptosis in AML, and engages a MYH9/β-catenin/c-Jun feedback loop to drive cancer stemness, with Septin9 serving as a negative upstream regulator that promotes EMT via FAK/Src signaling."},"narrative":{"mechanistic_narrative":"ARHGAP4 is a multi-domain Rho GTPase-activating protein that integrates cytoskeletal regulation with the control of cell and axon motility [PMID:12414125, PMID:17804252]. Recombinant ARHGAP4 stimulates the intrinsic GTPase activity of Rac1, Cdc42, and RhoA, establishing it as a functional GAP for multiple Rho-family members [PMID:12414125]. Its modular architecture partitions these activities: the FCH domain directs localization to the leading edges of migrating cells and to axon growth cones, while the GAP domain and C-terminus are required for its inhibition of cell migration and axon outgrowth [PMID:17804252]. Through its RhoGAP and SH3 domains, ARHGAP4 assembles a complex with the septins SEPT2 and SEPT9 that reorganizes focal adhesions and modulates integrin-β1–dependent migration and invasion [PMID:34524873], and it suppresses epithelial-to-mesenchymal transition and focal-adhesion–driven force generation, with Septin9 acting as a negative regulator that promotes EMT via FAK/Src signaling [PMID:32378260]. Beyond its canonical GAP role, ARHGAP4 functions as a scaffolding and regulatory protein in cancer: it promotes ubiquitination of HDAC2 to suppress β-catenin activation and tumor cell invasion [PMID:30958531], binds p53 to repress DRAM1-dependent apoptosis in acute myeloid leukemia [PMID:37443303], and drives colorectal cancer stemness through a MYH9/β-catenin/c-Jun feedback loop [PMID:40817404]. Its expression is suppressed by miR-939-5p [PMID:32021284].","teleology":[{"year":2002,"claim":"Established ARHGAP4 as a biochemically active GAP and placed it at the Golgi and motility structures, defining its candidate cellular context.","evidence":"In vitro GTPase assays with recombinant protein on Rac1/Cdc42/RhoA plus subcellular fractionation and immunofluorescence of endogenous protein","pmids":["12414125"],"confidence":"High","gaps":["In vitro substrate preference among Rac1/Cdc42/RhoA not quantified","Functional consequence of Golgi/microtubule localization unresolved"]},{"year":2007,"claim":"Mapped functional roles to specific domains, showing the FCH domain governs localization while the GAP domain and C-terminus drive motility inhibition.","evidence":"Domain-deletion structure/function analysis with live-cell migration and hippocampal axon outgrowth assays","pmids":["17804252"],"confidence":"Medium","gaps":["Which Rho GTPase mediates the migration/axon phenotype in cells not established","Endogenous loss-of-function not tested (overexpression-based)"]},{"year":2019,"claim":"Revealed a non-canonical scaffolding mechanism whereby ARHGAP4 promotes HDAC2 ubiquitination to restrain β-catenin and tumor invasion, and placed it upstream of the mTOR/HIF-1α Warburg axis.","evidence":"Co-IP, ubiquitination assay, siRNA knockdown, pharmacological rescue (CAY10683, XAV939, rapamycin, YC-1), invasion/migration and metabolic assays in pancreatic cancer cells","pmids":["30958531","31303760"],"confidence":"Medium","gaps":["Whether ARHGAP4 has direct E3 ligase activity or recruits one is not defined","Link between GAP activity and HDAC2/β-catenin axis unresolved"]},{"year":2020,"claim":"Identified upstream regulation by miR-939-5p and a septin-mediated route to EMT control, with Septin9 acting as a negative regulator via FAK/Src.","evidence":"Luciferase reporter target validation and rescue; RhoGAP siRNA screen, proteomics, traction force microscopy and 3D morphogenesis in mammary epithelial cells","pmids":["32021284","32378260"],"confidence":"Medium","gaps":["Mechanism by which Septin9 inhibits ARHGAP4 not defined at molecular level","Direct ARHGAP4–Septin9 binding interface not mapped here"]},{"year":2021,"claim":"Defined a direct ARHGAP4–SEPT2–SEPT9 complex and showed it bidirectionally controls integrin-β1–dependent focal adhesions and invasion.","evidence":"Reciprocal Co-IP with RhoGAP/SH3 domain mapping, siRNA knockdown, overexpression of multiple complex members, focal adhesion and migration/invasion assays","pmids":["34524873"],"confidence":"High","gaps":["Stoichiometry and structural architecture of the complex unknown","Whether GAP catalytic activity is required for focal adhesion remodeling not tested"]},{"year":2023,"claim":"Established a p53-binding, apoptosis-suppressing role in leukemia by placing ARHGAP4 upstream of p53/DRAM1.","evidence":"Co-IP (ARHGAP4-p53), siRNA knockdown, DRAM1 epistatic rescue, in vivo AML model and apoptosis/colony assays","pmids":["37443303"],"confidence":"Medium","gaps":["Binding interface and effect of ARHGAP4 on p53 transcriptional activity not detailed","Relationship between GAP function and p53 regulation unknown"]},{"year":2024,"claim":"Extended ARHGAP4's pro-tumor/EMT activity to colon cancer (TGF-β/Smad) and to apoptosis/inflammation in ovarian granulosa cells (PI3K-Akt).","evidence":"siRNA knockdown with Western blotting for pathway and EMT markers; CCK-8, annexin-V/PI, ELISA in granulosa cells and clinical tissue","pmids":["38805788","38355537"],"confidence":"Low","gaps":["No direct binding or rescue experiments to establish causality","Context-dependent opposite roles (pro- vs anti-tumor) not reconciled"]},{"year":2025,"claim":"Identified a MYH9/β-catenin/c-Jun positive feedback loop through which ARHGAP4 drives cancer stemness.","evidence":"Co-IP (ARHGAP4-MYH9), ChIP (c-Jun binding), FRAP, in vitro/in vivo stemness assays","pmids":["40817404"],"confidence":"Medium","gaps":["How the GAP/scaffolding domains engage MYH9 not mapped","Reconciliation with ARHGAP4's β-catenin-suppressing role in pancreatic cancer unresolved"]},{"year":null,"claim":"It remains unresolved how ARHGAP4's canonical Rho-GAP catalytic activity mechanistically connects to its diverse scaffolding functions (HDAC2 ubiquitination, p53, MYH9), and why it acts as a migration suppressor in some contexts but a tumor/stemness driver in others.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model linking GAP and scaffolding activities","Context-determinants of opposing phenotypes undefined","In vivo physiological (non-cancer) function not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,9,12]}],"complexes":["ARHGAP4–SEPT2–SEPT9 complex"],"partners":["SEPT2","SEPT9","HDAC2","TP53","MYH9"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P98171","full_name":"Rho GTPase-activating protein 4","aliases":["Rho-GAP hematopoietic protein C1","Rho-type GTPase-activating protein 4","p115"],"length_aa":946,"mass_kda":105.0,"function":"Inhibitory effect on stress fiber organization. May down-regulate Rho-like GTPase in hematopoietic cells","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P98171/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARHGAP4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ARHGAP4","total_profiled":1310},"omim":[{"mim_id":"618428","title":"POPOV-CHANG SYNDROME; POPCHAS","url":"https://www.omim.org/entry/618428"},{"mim_id":"601288","title":"TYROSINE 3-MONOOXYGENASE/TRYPTOPHAN 5-MONOOXYGENASE ACTIVATION PROTEIN, ZETA ISOFORM; YWHAZ","url":"https://www.omim.org/entry/601288"},{"mim_id":"300023","title":"RHO GTPase-ACTIVATING PROTEIN 4; ARHGAP4","url":"https://www.omim.org/entry/300023"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":106.5},{"tissue":"lymphoid tissue","ntpm":114.4}],"url":"https://www.proteinatlas.org/search/ARHGAP4"},"hgnc":{"alias_symbol":["KIAA0131","C1","p115","RhoGAP4","SrGAP4"],"prev_symbol":[]},"alphafold":{"accession":"P98171","domains":[{"cath_id":"1.20.1270.60","chopping":"2-78_97-197_221-282","consensus_level":"medium","plddt":92.2387,"start":2,"end":282},{"cath_id":"-","chopping":"349-397_430-482","consensus_level":"medium","plddt":88.9039,"start":349,"end":482},{"cath_id":"1.10.555.10","chopping":"516-695","consensus_level":"high","plddt":91.322,"start":516,"end":695},{"cath_id":"2.30.30.40","chopping":"750-802","consensus_level":"high","plddt":84.8147,"start":750,"end":802}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P98171","model_url":"https://alphafold.ebi.ac.uk/files/AF-P98171-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P98171-F1-predicted_aligned_error_v6.png","plddt_mean":75.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ARHGAP4","jax_strain_url":"https://www.jax.org/strain/search?query=ARHGAP4"},"sequence":{"accession":"P98171","fasta_url":"https://rest.uniprot.org/uniprotkb/P98171.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P98171/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P98171"}},"corpus_meta":[{"pmid":"17804252","id":"PMC_17804252","title":"ARHGAP4 is a novel RhoGAP that mediates inhibition of cell motility and axon outgrowth.","date":"2007","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/17804252","citation_count":55,"is_preprint":false},{"pmid":"31303760","id":"PMC_31303760","title":"ARHGAP4 mediates the Warburg effect in pancreatic cancer through the mTOR and HIF-1α signaling pathways.","date":"2019","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/31303760","citation_count":37,"is_preprint":false},{"pmid":"12414125","id":"PMC_12414125","title":"Cloning of rat ARHGAP4/C1, a RhoGAP family member expressed in the nervous system that colocalizes with the Golgi complex and microtubules.","date":"2002","source":"Brain research. Molecular brain research","url":"https://pubmed.ncbi.nlm.nih.gov/12414125","citation_count":35,"is_preprint":false},{"pmid":"11754100","id":"PMC_11754100","title":"Two novel types of contiguous gene deletion of the AVPR2 and ARHGAP4 genes in unrelated Japanese kindreds with nephrogenic diabetes insipidus.","date":"2002","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/11754100","citation_count":34,"is_preprint":false},{"pmid":"30958531","id":"PMC_30958531","title":"ARHGAP4 regulates the cell migration and invasion of pancreatic cancer by the HDAC2/β-catenin signaling pathway.","date":"2019","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/30958531","citation_count":33,"is_preprint":false},{"pmid":"32021284","id":"PMC_32021284","title":"miR-939-5p Contributes to the Migration and Invasion of Pancreatic Cancer by Targeting ARHGAP4.","date":"2020","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32021284","citation_count":24,"is_preprint":false},{"pmid":"32378260","id":"PMC_32378260","title":"Comprehensive analysis on the whole Rho-GAP family reveals that ARHGAP4 suppresses EMT in epithelial cells under negative regulation by Septin9.","date":"2020","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32378260","citation_count":24,"is_preprint":false},{"pmid":"34524873","id":"PMC_34524873","title":"ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions, cell migration, and invasion.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/34524873","citation_count":19,"is_preprint":false},{"pmid":"22965914","id":"PMC_22965914","title":"A novel contiguous gene deletion of AVPR2 and ARHGAP4 genes in male dizygotic twins with nephrogenic diabetes insipidus and intellectual disability.","date":"2012","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/22965914","citation_count":19,"is_preprint":false},{"pmid":"18489790","id":"PMC_18489790","title":"Immunological profile in a family with nephrogenic diabetes insipidus with a novel 11 kb deletion in AVPR2 and ARHGAP4 genes.","date":"2008","source":"BMC medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/18489790","citation_count":16,"is_preprint":false},{"pmid":"26707211","id":"PMC_26707211","title":"ARHGAP4 mutated in a Chinese intellectually challenged family.","date":"2015","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26707211","citation_count":8,"is_preprint":false},{"pmid":"37443303","id":"PMC_37443303","title":"ARHGAP4 promotes leukemogenesis in acute myeloid leukemia by inhibiting DRAM1 signaling.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/37443303","citation_count":7,"is_preprint":false},{"pmid":"36288877","id":"PMC_36288877","title":"Candidate Oncogenes, ARHGAP4, NOS3, and OR51B5, for the Development of Scirrhous-type Gastric Cancer.","date":"2022","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/36288877","citation_count":6,"is_preprint":false},{"pmid":"29394883","id":"PMC_29394883","title":"Contiguous 22.1-kb deletion embracing AVPR2 and ARHGAP4 genes at novel breakpoints leads to nephrogenic diabetes insipidus in a Chinese pedigree.","date":"2018","source":"BMC nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/29394883","citation_count":6,"is_preprint":false},{"pmid":"38805788","id":"PMC_38805788","title":"ARHGAP4 promotes colon cancer metastasis through the TGF-β signaling pathway and may be associated with T cell exhaustion.","date":"2024","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/38805788","citation_count":4,"is_preprint":false},{"pmid":"38938545","id":"PMC_38938545","title":"ARHGAP4 Inhibits Proliferation and Growth of SW620 Colon Cancer Cells by Cell Cycle and Differentiation Pathways.","date":"2024","source":"Scientifica","url":"https://pubmed.ncbi.nlm.nih.gov/38938545","citation_count":1,"is_preprint":false},{"pmid":"38355537","id":"PMC_38355537","title":"Cai's prescription inhibits granulosa cell apoptosis through ARHGAP4 on poor ovarian responders.","date":"2024","source":"Journal of ovarian research","url":"https://pubmed.ncbi.nlm.nih.gov/38355537","citation_count":1,"is_preprint":false},{"pmid":"40817404","id":"PMC_40817404","title":"ARHGAP4/MYH9/β-catenin/c-Jun positive feedback loop promotes colorectal cancer stemness.","date":"2025","source":"NPJ precision oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40817404","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11265,"output_tokens":3054,"usd":0.039802,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10495,"output_tokens":3226,"usd":0.066562,"stage2_stop_reason":"end_turn"},"total_usd":0.106364,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"Recombinant ARHGAP4 stimulates the GTPase activity of Rac1, Cdc42, and RhoA in vitro, establishing it as a functional GAP for multiple Rho family members.\",\n      \"method\": \"In vitro GTPase activity assay with recombinant protein\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic assay with recombinant protein on three substrates, single lab\",\n      \"pmids\": [\"12414125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Endogenous ARHGAP4 localizes to the Golgi complex and can redistribute to microtubules during mitosis; it is also detected at the tips of differentiating neurites in PC12 cells.\",\n      \"method\": \"Subcellular fractionation and immunofluorescence/localization studies\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization by direct imaging of endogenous protein, single lab, two cell contexts\",\n      \"pmids\": [\"12414125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The FCH domain of ARHGAP4 is required for localizing the protein to the leading edges of migrating NIH/3T3 cells and to axon growth cones, while the GAP domain and C-terminus are necessary for ARHGAP4-mediated inhibition of cell migration and axon outgrowth.\",\n      \"method\": \"Structure/function analysis with domain deletion constructs; live-cell migration assay and hippocampal axon outgrowth assay\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis combined with functional migration and axon outgrowth assays, single lab\",\n      \"pmids\": [\"17804252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Overexpression of ARHGAP4 inhibits NIH/3T3 cell migration and hippocampal axon outgrowth, establishing a functional role as an inhibitor of cell and axon motility.\",\n      \"method\": \"Overexpression in NIH/3T3 cells (migration assay) and hippocampal neurons (axon outgrowth assay)\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined loss/gain-of-function with specific phenotypic readouts, single lab\",\n      \"pmids\": [\"17804252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ARHGAP4 interacts with and promotes ubiquitination of HDAC2, which in turn inhibits β-catenin activation, suppressing MMP2/MMP9 expression and pancreatic cancer cell invasion and migration.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, pharmacological inhibitors (CAY10683, XAV939), invasion/migration assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional rescue with inhibitors, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30958531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ARHGAP4 overexpression inhibits cell viability, glucose uptake, lactate release, PKM2 expression, and mTOR/HIF-1α pathway activation in pancreatic cancer cells; mTOR inhibitor (rapamycin) or HIF-1α inhibitor (YC-1) rescues the morphological changes induced by ARHGAP4 downregulation, placing ARHGAP4 upstream of the mTOR/HIF-1α axis in the Warburg effect.\",\n      \"method\": \"Lentiviral overexpression and siRNA knockdown; pharmacological inhibition of mTOR and HIF-1α; glucose uptake and lactate release assays; Western blotting\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via pharmacological rescue combined with gain/loss-of-function, single lab\",\n      \"pmids\": [\"31303760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-939-5p directly targets and suppresses ARHGAP4 expression (validated by luciferase reporter assay), thereby promoting pancreatic cancer cell viability, invasion, and migration; ARHGAP4 overexpression reverses these effects.\",\n      \"method\": \"Luciferase reporter assay, RT-PCR, Western blot, Transwell invasion/migration assay, rescue experiment\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct target validation by luciferase reporter plus functional rescue, single lab\",\n      \"pmids\": [\"32021284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ARHGAP4 suppresses epithelial-to-mesenchymal transition (EMT) in human mammary epithelial cells, regulating epithelial/mesenchymal marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven force generation; Septin9 was identified by proteomics as a negative regulator of ARHGAP4 that promotes EMT via FAK/Src signaling.\",\n      \"method\": \"Comprehensive RhoGAP siRNA screen, proteomic analysis, cell morphology/migration assays, traction force microscopy, 3D morphogenesis assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multidisciplinary functional assays plus proteomic identification of binding partner, single lab\",\n      \"pmids\": [\"32378260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ARHGAP4 forms a complex with SEPT2 and SEPT9 via its RhoGAP domain and SH3 domain; silencing ARHGAP4 or overexpressing SEPT2/SEPT9 independently induces reorganization of focal adhesions with upregulation of Integrin Beta 1, enhancing cell migration and invasion in a microenvironment-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, siRNA knockdown, overexpression, focal adhesion analysis, migration/invasion assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with domain mapping plus multiple orthogonal functional assays, replicated across perturbations (KD and OE of multiple complex members)\",\n      \"pmids\": [\"34524873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ARHGAP4 binds p53 to inhibit DRAM1 expression in AML cells; ARHGAP4 knockdown activates DRAM1 signaling and induces apoptosis, while DRAM1 knockdown rescues the defects caused by ARHGAP4 deletion, placing ARHGAP4 upstream of p53/DRAM1 in AML leukemogenesis.\",\n      \"method\": \"Co-immunoprecipitation (ARHGAP4-p53 interaction), siRNA knockdown, in vivo AML progression model, apoptosis assay, colony formation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus genetic epistasis (DRAM1 KD rescue), in vivo validation, single lab\",\n      \"pmids\": [\"37443303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARHGAP4 knockdown inhibits migration and invasion of colon cancer cells and decreases expression of TGF-β1, p-Smad2, and p-Smad3, while increasing E-cadherin and decreasing N-cadherin/Vimentin, placing ARHGAP4 upstream of the TGF-β/Smad pathway and EMT in colon cancer.\",\n      \"method\": \"siRNA knockdown, Western blotting for TGF-β/Smad pathway proteins and EMT markers, Transwell migration/invasion assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single set of methods, no direct binding or rescue experiments\",\n      \"pmids\": [\"38805788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARHGAP4 promotes apoptosis and inflammation in ovarian granulosa cells via the PI3K-Akt signaling pathway.\",\n      \"method\": \"CCK-8 assay, annexin-V/PI staining, ELISA, Western blot for PI3K-Akt pathway components, cell line and clinical tissue samples\",\n      \"journal\": \"Journal of ovarian research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited mechanistic depth, no direct binding or epistasis experiments\",\n      \"pmids\": [\"38355537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ARHGAP4 drives colorectal cancer stemness through a positive feedback loop with MYH9/β-catenin/c-Jun, as demonstrated by co-immunoprecipitation (ARHGAP4-MYH9 interaction), chromatin immunoprecipitation (c-Jun binding), and FRAP.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation, FRAP, in vitro and in vivo stemness assays, bioinformatics\",\n      \"journal\": \"NPJ precision oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, ChIP, FRAP) in single lab study\",\n      \"pmids\": [\"40817404\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGAP4 is a multi-domain RhoGAP (FCH–GAP–SH3) that stimulates GTPase activity of Rac1, Cdc42, and RhoA; its FCH domain targets it to leading edges and growth cones where it inhibits cell migration and axon outgrowth, while its GAP and SH3 domains mediate assembly of an ARHGAP4–SEPT2–SEPT9 complex that bidirectionally modulates integrin-β1-dependent focal adhesions; in addition to its canonical GAP function, ARHGAP4 acts as a scaffolding/regulatory protein that ubiquitinates HDAC2 to suppress β-catenin/Wnt signaling, binds p53 to repress DRAM1-dependent apoptosis in AML, and engages a MYH9/β-catenin/c-Jun feedback loop to drive cancer stemness, with Septin9 serving as a negative upstream regulator that promotes EMT via FAK/Src signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ARHGAP4 is a multi-domain Rho GTPase-activating protein that integrates cytoskeletal regulation with the control of cell and axon motility [#0, #3]. Recombinant ARHGAP4 stimulates the intrinsic GTPase activity of Rac1, Cdc42, and RhoA, establishing it as a functional GAP for multiple Rho-family members [#0]. Its modular architecture partitions these activities: the FCH domain directs localization to the leading edges of migrating cells and to axon growth cones, while the GAP domain and C-terminus are required for its inhibition of cell migration and axon outgrowth [#2, #3]. Through its RhoGAP and SH3 domains, ARHGAP4 assembles a complex with the septins SEPT2 and SEPT9 that reorganizes focal adhesions and modulates integrin-\\u03b21\\u2013dependent migration and invasion [#8], and it suppresses epithelial-to-mesenchymal transition and focal-adhesion\\u2013driven force generation, with Septin9 acting as a negative regulator that promotes EMT via FAK/Src signaling [#7]. Beyond its canonical GAP role, ARHGAP4 functions as a scaffolding and regulatory protein in cancer: it promotes ubiquitination of HDAC2 to suppress \\u03b2-catenin activation and tumor cell invasion [#4], binds p53 to repress DRAM1-dependent apoptosis in acute myeloid leukemia [#9], and drives colorectal cancer stemness through a MYH9/\\u03b2-catenin/c-Jun feedback loop [#12]. Its expression is suppressed by miR-939-5p [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established ARHGAP4 as a biochemically active GAP and placed it at the Golgi and motility structures, defining its candidate cellular context.\",\n      \"evidence\": \"In vitro GTPase assays with recombinant protein on Rac1/Cdc42/RhoA plus subcellular fractionation and immunofluorescence of endogenous protein\",\n      \"pmids\": [\"12414125\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro substrate preference among Rac1/Cdc42/RhoA not quantified\", \"Functional consequence of Golgi/microtubule localization unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped functional roles to specific domains, showing the FCH domain governs localization while the GAP domain and C-terminus drive motility inhibition.\",\n      \"evidence\": \"Domain-deletion structure/function analysis with live-cell migration and hippocampal axon outgrowth assays\",\n      \"pmids\": [\"17804252\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which Rho GTPase mediates the migration/axon phenotype in cells not established\", \"Endogenous loss-of-function not tested (overexpression-based)\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a non-canonical scaffolding mechanism whereby ARHGAP4 promotes HDAC2 ubiquitination to restrain \\u03b2-catenin and tumor invasion, and placed it upstream of the mTOR/HIF-1\\u03b1 Warburg axis.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, siRNA knockdown, pharmacological rescue (CAY10683, XAV939, rapamycin, YC-1), invasion/migration and metabolic assays in pancreatic cancer cells\",\n      \"pmids\": [\"30958531\", \"31303760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ARHGAP4 has direct E3 ligase activity or recruits one is not defined\", \"Link between GAP activity and HDAC2/\\u03b2-catenin axis unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified upstream regulation by miR-939-5p and a septin-mediated route to EMT control, with Septin9 acting as a negative regulator via FAK/Src.\",\n      \"evidence\": \"Luciferase reporter target validation and rescue; RhoGAP siRNA screen, proteomics, traction force microscopy and 3D morphogenesis in mammary epithelial cells\",\n      \"pmids\": [\"32021284\", \"32378260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Septin9 inhibits ARHGAP4 not defined at molecular level\", \"Direct ARHGAP4\\u2013Septin9 binding interface not mapped here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a direct ARHGAP4\\u2013SEPT2\\u2013SEPT9 complex and showed it bidirectionally controls integrin-\\u03b21\\u2013dependent focal adhesions and invasion.\",\n      \"evidence\": \"Reciprocal Co-IP with RhoGAP/SH3 domain mapping, siRNA knockdown, overexpression of multiple complex members, focal adhesion and migration/invasion assays\",\n      \"pmids\": [\"34524873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural architecture of the complex unknown\", \"Whether GAP catalytic activity is required for focal adhesion remodeling not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a p53-binding, apoptosis-suppressing role in leukemia by placing ARHGAP4 upstream of p53/DRAM1.\",\n      \"evidence\": \"Co-IP (ARHGAP4-p53), siRNA knockdown, DRAM1 epistatic rescue, in vivo AML model and apoptosis/colony assays\",\n      \"pmids\": [\"37443303\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface and effect of ARHGAP4 on p53 transcriptional activity not detailed\", \"Relationship between GAP function and p53 regulation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended ARHGAP4's pro-tumor/EMT activity to colon cancer (TGF-\\u03b2/Smad) and to apoptosis/inflammation in ovarian granulosa cells (PI3K-Akt).\",\n      \"evidence\": \"siRNA knockdown with Western blotting for pathway and EMT markers; CCK-8, annexin-V/PI, ELISA in granulosa cells and clinical tissue\",\n      \"pmids\": [\"38805788\", \"38355537\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct binding or rescue experiments to establish causality\", \"Context-dependent opposite roles (pro- vs anti-tumor) not reconciled\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a MYH9/\\u03b2-catenin/c-Jun positive feedback loop through which ARHGAP4 drives cancer stemness.\",\n      \"evidence\": \"Co-IP (ARHGAP4-MYH9), ChIP (c-Jun binding), FRAP, in vitro/in vivo stemness assays\",\n      \"pmids\": [\"40817404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How the GAP/scaffolding domains engage MYH9 not mapped\", \"Reconciliation with ARHGAP4's \\u03b2-catenin-suppressing role in pancreatic cancer unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how ARHGAP4's canonical Rho-GAP catalytic activity mechanistically connects to its diverse scaffolding functions (HDAC2 ubiquitination, p53, MYH9), and why it acts as a migration suppressor in some contexts but a tumor/stemness driver in others.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model linking GAP and scaffolding activities\", \"Context-determinants of opposing phenotypes undefined\", \"In vivo physiological (non-cancer) function not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 9, 12]}\n    ],\n    \"complexes\": [\"ARHGAP4\\u2013SEPT2\\u2013SEPT9 complex\"],\n    \"partners\": [\"SEPT2\", \"SEPT9\", \"HDAC2\", \"TP53\", \"MYH9\", \"SEPT9\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}