{"gene":"NHSL3","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2025,"finding":"NHSL3 knockout cells show increased persistence in single cell migration but impaired collective migration (follower cells cannot follow leader cells in wound healing), demonstrating distinct roles in single vs. collective migration.","method":"Knockout cell lines, wound healing assay, single cell migration tracking","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal rescue experiments with specific isoforms and binding-site mutants in a peer-reviewed study; multiple orthogonal methods","pmids":["39747206"],"is_preprint":false},{"year":2025,"finding":"A long isoform of NHSL3 interacts with MENA/VASP proteins at cell-cell junctions and this interaction is required for collective cell migration; mutation of the relevant binding sites abolishes rescue.","method":"Proteomics (isoform-specific interactome), AlphaFold2-based binding site prediction, isoform-specific rescue experiments, binding-site mutagenesis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — isoform-specific rescue with binding-site mutagenesis plus proteomics, single rigorous study with multiple orthogonal methods","pmids":["39747206"],"is_preprint":false},{"year":2025,"finding":"A short isoform of NHSL3 interacts with 14-3-3θ in lamellipodia and this interaction is required for single cell migration persistence; mutation of the relevant binding sites abolishes rescue.","method":"Proteomics (isoform-specific interactome), AlphaFold2-based binding site prediction, isoform-specific rescue experiments, binding-site mutagenesis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — isoform-specific rescue with binding-site mutagenesis plus proteomics, single rigorous study with multiple orthogonal methods","pmids":["39747206"],"is_preprint":false},{"year":2025,"finding":"NHSL3 co-localizes at the edge of lamellipodia with Ena/VASP proteins and the Scar/WAVE complex, promotes cell migration, and functions to inhibit Scar/WAVE-Arp2/3 activity in cells.","method":"Co-localization imaging, co-immunoprecipitation/pulldown, functional migration assays, Arp2/3 activity assay in cells","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and co-localization shown, functional inhibition of Arp2/3 demonstrated, but preprint and single lab","pmids":["bio_10.1101_2025.04.03.647056"],"is_preprint":true},{"year":2025,"finding":"NHSL3 binds to the Scar/WAVE complex subunit Abi and, uniquely among known Scar/WAVE complex binders, also binds to CYFIP1/2 through three short linear motifs.","method":"Pulldown/binding assays, short linear motif mapping","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding shown with motif mapping, preprint and single lab","pmids":["bio_10.1101_2025.04.03.647056"],"is_preprint":true},{"year":2017,"finding":"siRNA-mediated silencing of KIAA1522 in esophageal squamous cell carcinoma cells markedly reduced phosphorylated ERK levels in both suspended and adherent cells, indicating KIAA1522 promotes cell proliferation and anoikis resistance via ERK cascade activation.","method":"siRNA knockdown, Western blot for phospho-ERK, in vitro proliferation and anoikis resistance assays, xenograft tumor and lung metastasis models","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined molecular readout (p-ERK), in vivo validation, single lab","pmids":["28794639"],"is_preprint":false},{"year":2020,"finding":"KIAA1522 potentiates TNFα-TNFR2-NFκB signaling in lung adenocarcinoma cells, promoting resistance to cisplatin; NFκB inhibition abolishes the KIAA1522-driven cisplatin resistance in vivo.","method":"In vivo KrasG12D/Cas9 mouse model with AAV-mediated sgRNA KIAA1522 depletion, pharmacological NFκB inhibition, in vitro cisplatin assays","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic depletion model with pathway inhibitor rescue, single lab with multiple methods","pmids":["32854746"],"is_preprint":false},{"year":2020,"finding":"KIAA1522 upregulates the Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells, as shown by TOP-flash/FOP-flash luciferase reporter assays.","method":"TOP-flash/FOP-flash luciferase reporter assay, Western blot, knockdown/overexpression functional assays","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reporter assay plus functional assays showing pathway activation, single lab","pmids":["32606779"],"is_preprint":false},{"year":2021,"finding":"KIAA1522 upregulates the Notch signaling pathway in colorectal cancer cells in vitro and in lung metastatic nodes in vivo, promoting proliferation, invasion, migration, and distant metastasis.","method":"Transcriptome sequencing, GSEA, Western blot, IHC, tail vein injection in vivo model, functional assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — transcriptome plus in vivo validation, single lab","pmids":["34826587"],"is_preprint":false},{"year":2024,"finding":"Transcription factor KLF9 binds the KIAA1522 promoter and represses KIAA1522 expression, thereby inhibiting pancreatic cancer cell proliferation, invasion, and migration; KIAA1522 overexpression rescues the KLF9-mediated inhibition.","method":"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), overexpression/knockdown rescue experiments","journal":"Asia-Pacific journal of clinical oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus luciferase reporter with rescue experiment, single lab","pmids":["38520660"],"is_preprint":false},{"year":2025,"finding":"LncRNA PLACT1 recruits EHMT2 to induce H3K9me2 at the KLF2 promoter, repressing KLF2 expression, which reduces KLF2 enrichment at the KIAA1522 promoter, thereby increasing KIAA1522 expression and promoting pancreatic adenocarcinoma cell proliferation.","method":"ChIP assay for EHMT2 and H3K9me2, luciferase reporter for KLF2-KIAA1522 promoter binding, RNA pulldown for PLACT1-EHMT2, rescue experiments, nude mouse xenograft","journal":"Histology and histopathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, pulldown, and in vivo model, single lab with multiple orthogonal methods","pmids":["41164921"],"is_preprint":false},{"year":2025,"finding":"KIAA1522 has state-specific isoforms in human ESCs regulated by alternative first exon usage; the naïve-specific isoform and primed-specific isoform have distinct N-terminal domains and different subcellular localizations, and have opposite effects on LTR retrotransposon activity.","method":"RNA sequencing, CUT&Tag with isoform-specific overexpression or knockdown, subcellular localization imaging","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CUT&Tag with isoform-specific manipulation and localization, single lab with multiple methods","pmids":["40553414"],"is_preprint":false},{"year":2022,"finding":"HOXA-AS2 recruits IGF2BP3 to stabilize KIAA1522 mRNA, providing a post-transcriptional mechanism for KIAA1522 upregulation in vascular smooth muscle cells.","method":"RNA immunoprecipitation (RIP), luciferase reporter, RT-qPCR, Western blot, functional assays","journal":"ESC heart failure","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, RIP assay without full mechanistic reconstitution, limited follow-up on KIAA1522 itself","pmids":["35730141"],"is_preprint":false}],"current_model":"NHSL3/KIAA1522 is a multi-isoform protein belonging to the Nance-Horan Syndrome family that regulates cell migration through at least two distinct isoform-specific mechanisms: a long isoform interacts with MENA/VASP proteins at cell-cell junctions to enable collective migration, while a short isoform interacts with 14-3-3θ in lamellipodia to modulate single-cell migration persistence; NHSL3 also co-localizes with and inhibits Scar/WAVE-Arp2/3 activity at lamellipodia by binding both the Abi and CYFIP1/2 subunits of the Scar/WAVE complex via short linear motifs; additionally, KIAA1522 promotes tumor cell proliferation and survival through activation of ERK, NFκB, Wnt/β-catenin, and Notch signaling pathways, and its expression is transcriptionally repressed by KLF9 and regulated epigenetically via the PLACT1-EHMT2-KLF2 axis, while isoform-specific nuclear functions regulate LTR retrotransposon activity in distinct pluripotency states."},"narrative":{"mechanistic_narrative":"NHSL3 (KIAA1522) is a multi-isoform member of the Nance-Horan Syndrome family that regulates actin-based cell migration through isoform-specific protein interactions at the leading edge and at cell-cell junctions [PMID:39747206]. Loss of NHSL3 increases persistence of single-cell migration while impairing collective migration, reflecting two genetically separable functions [PMID:39747206]: a long isoform binds MENA/VASP proteins at cell-cell junctions to enable collective migration, whereas a short isoform binds 14-3-3θ in lamellipodia to set single-cell migration persistence, with binding-site mutations abolishing each rescue [PMID:39747206]. At the lamellipodial edge NHSL3 co-localizes with Ena/VASP and the Scar/WAVE complex and inhibits Scar/WAVE-Arp2/3 activity [PMID:bio_10.1101_2025.04.03.647056], engaging the complex through both its Abi and CYFIP1/2 subunits via three short linear motifs [PMID:bio_10.1101_2025.04.03.647056]. Beyond motility, KIAA1522 promotes tumor cell proliferation, survival, and chemoresistance by activating ERK [PMID:28794639], TNFα-TNFR2-NFκB [PMID:32854746], Wnt/β-catenin [PMID:32606779], and Notch [PMID:34826587] signaling. Its expression is controlled transcriptionally by KLF9, which binds the KIAA1522 promoter and represses it [PMID:38520660], and epigenetically through a PLACT1-EHMT2-KLF2 axis that de-represses KIAA1522 [PMID:41164921]; alternative first-exon usage generates state-specific isoforms in pluripotent cells that differ in N-terminal domain, localization, and effect on LTR retrotransposon activity [PMID:40553414].","teleology":[{"year":2017,"claim":"Established the first molecular handle on KIAA1522 function by linking it to a defined growth-signaling readout, addressing why its expression correlates with tumor aggressiveness.","evidence":"siRNA knockdown with phospho-ERK Western blot, proliferation/anoikis assays, and xenograft/metastasis models in esophageal squamous cell carcinoma","pmids":["28794639"],"confidence":"Medium","gaps":["Does not show direct molecular interaction between KIAA1522 and ERK pathway components","Mechanism of ERK activation unresolved"]},{"year":2020,"claim":"Extended KIAA1522's pro-tumor role to additional survival pathways, showing it potentiates NFκB and Wnt/β-catenin signaling and drives chemoresistance.","evidence":"In vivo KrasG12D/Cas9 model with AAV-sgRNA depletion plus NFκB inhibitor rescue (lung adenocarcinoma); TOP/FOP-flash luciferase reporters (hepatocellular carcinoma)","pmids":["32854746","32606779"],"confidence":"Medium","gaps":["Direct binding partners connecting KIAA1522 to NFκB/Wnt not identified","Whether pathway activation is direct or secondary unknown"]},{"year":2021,"claim":"Added Notch as a fourth oncogenic pathway downstream of KIAA1522, reinforcing its role in proliferation, invasion, and metastasis.","evidence":"Transcriptome sequencing, GSEA, Western blot, IHC and tail-vein metastasis model in colorectal cancer","pmids":["34826587"],"confidence":"Medium","gaps":["Mechanism linking KIAA1522 to Notch activation not defined","No physical interaction demonstrated"]},{"year":2024,"claim":"Answered how KIAA1522 levels are set transcriptionally, identifying KLF9 as a direct promoter-binding repressor.","evidence":"ChIP and dual-luciferase reporter with overexpression/knockdown rescue in pancreatic cancer","pmids":["38520660"],"confidence":"Medium","gaps":["Upstream regulators of KLF9 in this context not defined","Single tumor type"]},{"year":2025,"claim":"Resolved the core cell-biological function of NHSL3, demonstrating isoform-specific interactions that partition single-cell versus collective migration control.","evidence":"Knockout cell lines, wound healing and single-cell tracking, isoform-specific proteomics, AlphaFold2 binding prediction, and binding-site mutagenesis rescue (Nature Communications)","pmids":["39747206"],"confidence":"High","gaps":["Structural basis of the MENA/VASP and 14-3-3θ interactions not determined","How isoform choice is regulated across tissues unknown"]},{"year":2025,"claim":"Defined a molecular mechanism for NHSL3's effect on the actin machinery by showing it binds and inhibits the Scar/WAVE-Arp2/3 system.","evidence":"Co-localization imaging, pulldown/Co-IP, short linear motif mapping, and cellular Arp2/3 activity assay (bioRxiv preprint)","pmids":["bio_10.1101_2025.04.03.647056"],"confidence":"Medium","gaps":["Preprint, single lab","In vitro reconstitution of Arp2/3 inhibition not shown","Functional consequence of dual Abi/CYFIP binding unresolved"]},{"year":2025,"claim":"Uncovered an epigenetic axis and a nuclear, isoform-dependent function, broadening KIAA1522 beyond cytoplasmic migration into transcriptional/chromatin contexts.","evidence":"ChIP/RNA pulldown/xenograft for the PLACT1-EHMT2-KLF2 axis; RNA-seq, CUT&Tag and localization for state-specific ESC isoforms regulating LTR retrotransposons","pmids":["41164921","40553414"],"confidence":"Medium","gaps":["Direct chromatin-binding activity of nuclear NHSL3 isoforms not characterized","Mechanism by which isoforms affect LTR activity unknown","Single lab per finding"]},{"year":null,"claim":"How the distinct isoform-specific cytoplasmic (migration), oncogenic signaling, and nuclear (retrotransposon) functions are coordinated within and across cell types remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying biochemical activity defined for the protein","Relationship between migration role and oncogenic signaling not established","No structural model of the protein"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,3,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,4]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7,8]}],"complexes":[],"partners":["VASP","ENAH","YWHAQ","ABI1","CYFIP1","CYFIP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P206","full_name":"NHS-like protein 3","aliases":[],"length_aa":1035,"mass_kda":107.1,"function":"Able to directly activate the TNF-NFkappaB signaling pathway","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9P206/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NHSL3","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"NCKAP1","stoichiometry":10.0},{"gene":"ACTG1","stoichiometry":0.2},{"gene":"BAIAP2","stoichiometry":0.2},{"gene":"CSNK1G3","stoichiometry":0.2},{"gene":"PFN1","stoichiometry":0.2},{"gene":"PKMYT1","stoichiometry":0.2},{"gene":"RBM8A","stoichiometry":0.2},{"gene":"SRP68","stoichiometry":0.2},{"gene":"WASF1","stoichiometry":0.2},{"gene":"WASF2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NHSL3","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"KIAA1522","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cell Junctions","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KIAA1522"},"hgnc":{"alias_symbol":[],"prev_symbol":["KIAA1522"]},"alphafold":{"accession":"Q9P206","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P206","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P206-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P206-F1-predicted_aligned_error_v6.png","plddt_mean":48.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NHSL3","jax_strain_url":"https://www.jax.org/strain/search?query=NHSL3"},"sequence":{"accession":"Q9P206","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P206.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P206/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P206"}},"corpus_meta":[{"pmid":"30177391","id":"PMC_30177391","title":"miR-125b-5p inhibits breast cancer cell proliferation, migration and invasion by targeting KIAA1522.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30177391","citation_count":82,"is_preprint":false},{"pmid":"33318318","id":"PMC_33318318","title":"LncRNA CYTOR affects the proliferation, cell cycle and apoptosis of hepatocellular carcinoma cells by regulating the miR-125b-5p/KIAA1522 axis.","date":"2020","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/33318318","citation_count":29,"is_preprint":false},{"pmid":"33794727","id":"PMC_33794727","title":"LncRNA FGD5-AS1 accelerates cell proliferation in pancreatic cancer by regulating miR-520a-3p/KIAA1522 axis.","date":"2021","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33794727","citation_count":26,"is_preprint":false},{"pmid":"28794639","id":"PMC_28794639","title":"KIAA1522 overexpression promotes tumorigenicity and metastasis of esophageal cancer cells through potentiating the ERK activity.","date":"2017","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/28794639","citation_count":24,"is_preprint":false},{"pmid":"36528986","id":"PMC_36528986","title":"Exosome-mediated circTTLL5 transfer promotes hepatocellular carcinoma malignant progression through miR-136-5p/KIAA1522 axis.","date":"2022","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/36528986","citation_count":17,"is_preprint":false},{"pmid":"32854746","id":"PMC_32854746","title":"KIAA1522 potentiates TNFα-NFκB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma.","date":"2020","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/32854746","citation_count":16,"is_preprint":false},{"pmid":"33122952","id":"PMC_33122952","title":"Long Non-Coding RNA USP2-AS1 Accelerates Cell Proliferation and Migration in Ovarian Cancer by Sponging miR-520d-3p and Up-Regulating KIAA1522.","date":"2020","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/33122952","citation_count":14,"is_preprint":false},{"pmid":"32606779","id":"PMC_32606779","title":"KIAA1522 Promotes the Progression of Hepatocellular Carcinoma via the Activation of the Wnt/β-Catenin Signaling Pathway.","date":"2020","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32606779","citation_count":12,"is_preprint":false},{"pmid":"34826587","id":"PMC_34826587","title":"KIAA1522 is a new biomarker of promoting the tumorigenesis and distant metastasis of colorectal carcinoma.","date":"2021","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/34826587","citation_count":11,"is_preprint":false},{"pmid":"35196193","id":"PMC_35196193","title":"Circ_0020123 promotes NSCLC tumorigenesis via up-regulating KIAA1522 expression through miR-940.","date":"2022","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/35196193","citation_count":8,"is_preprint":false},{"pmid":"35730141","id":"PMC_35730141","title":"HOXA cluster antisense RNA 2 elevates KIAA1522 expression through microRNA-520d-3p and insulin like growth factor 2 mRNA binding protein 3 to promote the growth of vascular smooth muscle cells in thoracic aortic aneurysm.","date":"2022","source":"ESC heart failure","url":"https://pubmed.ncbi.nlm.nih.gov/35730141","citation_count":6,"is_preprint":false},{"pmid":"38520660","id":"PMC_38520660","title":"Transcription factor KLF9 inhibits the proliferation, invasion, and migration of pancreatic cancer cells by repressing KIAA1522.","date":"2024","source":"Asia-Pacific journal of clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38520660","citation_count":5,"is_preprint":false},{"pmid":"39747206","id":"PMC_39747206","title":"NHSL3 controls single and collective cell migration through two distinct mechanisms.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39747206","citation_count":2,"is_preprint":false},{"pmid":"40553414","id":"PMC_40553414","title":"KIAA1522 isoform switching regulates LTR-RTs activity in distinct pluripotency states of hESCs.","date":"2025","source":"Science China. Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40553414","citation_count":1,"is_preprint":false},{"pmid":"41164921","id":"PMC_41164921","title":"Mechanism of lncRNA PLACT1 in regulating the proliferation of pancreatic adenocarcinoma cells through the KLF2/KIAA1522 axis.","date":"2025","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/41164921","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.03.647056","title":"NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration","date":"2025-04-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.03.647056","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10601,"output_tokens":2959,"usd":0.038094,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10324,"output_tokens":2803,"usd":0.060847,"stage2_stop_reason":"end_turn"},"total_usd":0.098941,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2025,\n      \"finding\": \"NHSL3 knockout cells show increased persistence in single cell migration but impaired collective migration (follower cells cannot follow leader cells in wound healing), demonstrating distinct roles in single vs. collective migration.\",\n      \"method\": \"Knockout cell lines, wound healing assay, single cell migration tracking\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal rescue experiments with specific isoforms and binding-site mutants in a peer-reviewed study; multiple orthogonal methods\",\n      \"pmids\": [\"39747206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A long isoform of NHSL3 interacts with MENA/VASP proteins at cell-cell junctions and this interaction is required for collective cell migration; mutation of the relevant binding sites abolishes rescue.\",\n      \"method\": \"Proteomics (isoform-specific interactome), AlphaFold2-based binding site prediction, isoform-specific rescue experiments, binding-site mutagenesis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — isoform-specific rescue with binding-site mutagenesis plus proteomics, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"39747206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A short isoform of NHSL3 interacts with 14-3-3θ in lamellipodia and this interaction is required for single cell migration persistence; mutation of the relevant binding sites abolishes rescue.\",\n      \"method\": \"Proteomics (isoform-specific interactome), AlphaFold2-based binding site prediction, isoform-specific rescue experiments, binding-site mutagenesis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — isoform-specific rescue with binding-site mutagenesis plus proteomics, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"39747206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NHSL3 co-localizes at the edge of lamellipodia with Ena/VASP proteins and the Scar/WAVE complex, promotes cell migration, and functions to inhibit Scar/WAVE-Arp2/3 activity in cells.\",\n      \"method\": \"Co-localization imaging, co-immunoprecipitation/pulldown, functional migration assays, Arp2/3 activity assay in cells\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and co-localization shown, functional inhibition of Arp2/3 demonstrated, but preprint and single lab\",\n      \"pmids\": [\"bio_10.1101_2025.04.03.647056\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NHSL3 binds to the Scar/WAVE complex subunit Abi and, uniquely among known Scar/WAVE complex binders, also binds to CYFIP1/2 through three short linear motifs.\",\n      \"method\": \"Pulldown/binding assays, short linear motif mapping\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding shown with motif mapping, preprint and single lab\",\n      \"pmids\": [\"bio_10.1101_2025.04.03.647056\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"siRNA-mediated silencing of KIAA1522 in esophageal squamous cell carcinoma cells markedly reduced phosphorylated ERK levels in both suspended and adherent cells, indicating KIAA1522 promotes cell proliferation and anoikis resistance via ERK cascade activation.\",\n      \"method\": \"siRNA knockdown, Western blot for phospho-ERK, in vitro proliferation and anoikis resistance assays, xenograft tumor and lung metastasis models\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined molecular readout (p-ERK), in vivo validation, single lab\",\n      \"pmids\": [\"28794639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KIAA1522 potentiates TNFα-TNFR2-NFκB signaling in lung adenocarcinoma cells, promoting resistance to cisplatin; NFκB inhibition abolishes the KIAA1522-driven cisplatin resistance in vivo.\",\n      \"method\": \"In vivo KrasG12D/Cas9 mouse model with AAV-mediated sgRNA KIAA1522 depletion, pharmacological NFκB inhibition, in vitro cisplatin assays\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic depletion model with pathway inhibitor rescue, single lab with multiple methods\",\n      \"pmids\": [\"32854746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KIAA1522 upregulates the Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells, as shown by TOP-flash/FOP-flash luciferase reporter assays.\",\n      \"method\": \"TOP-flash/FOP-flash luciferase reporter assay, Western blot, knockdown/overexpression functional assays\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reporter assay plus functional assays showing pathway activation, single lab\",\n      \"pmids\": [\"32606779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"KIAA1522 upregulates the Notch signaling pathway in colorectal cancer cells in vitro and in lung metastatic nodes in vivo, promoting proliferation, invasion, migration, and distant metastasis.\",\n      \"method\": \"Transcriptome sequencing, GSEA, Western blot, IHC, tail vein injection in vivo model, functional assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — transcriptome plus in vivo validation, single lab\",\n      \"pmids\": [\"34826587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Transcription factor KLF9 binds the KIAA1522 promoter and represses KIAA1522 expression, thereby inhibiting pancreatic cancer cell proliferation, invasion, and migration; KIAA1522 overexpression rescues the KLF9-mediated inhibition.\",\n      \"method\": \"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), overexpression/knockdown rescue experiments\",\n      \"journal\": \"Asia-Pacific journal of clinical oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus luciferase reporter with rescue experiment, single lab\",\n      \"pmids\": [\"38520660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LncRNA PLACT1 recruits EHMT2 to induce H3K9me2 at the KLF2 promoter, repressing KLF2 expression, which reduces KLF2 enrichment at the KIAA1522 promoter, thereby increasing KIAA1522 expression and promoting pancreatic adenocarcinoma cell proliferation.\",\n      \"method\": \"ChIP assay for EHMT2 and H3K9me2, luciferase reporter for KLF2-KIAA1522 promoter binding, RNA pulldown for PLACT1-EHMT2, rescue experiments, nude mouse xenograft\",\n      \"journal\": \"Histology and histopathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, pulldown, and in vivo model, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41164921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KIAA1522 has state-specific isoforms in human ESCs regulated by alternative first exon usage; the naïve-specific isoform and primed-specific isoform have distinct N-terminal domains and different subcellular localizations, and have opposite effects on LTR retrotransposon activity.\",\n      \"method\": \"RNA sequencing, CUT&Tag with isoform-specific overexpression or knockdown, subcellular localization imaging\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CUT&Tag with isoform-specific manipulation and localization, single lab with multiple methods\",\n      \"pmids\": [\"40553414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HOXA-AS2 recruits IGF2BP3 to stabilize KIAA1522 mRNA, providing a post-transcriptional mechanism for KIAA1522 upregulation in vascular smooth muscle cells.\",\n      \"method\": \"RNA immunoprecipitation (RIP), luciferase reporter, RT-qPCR, Western blot, functional assays\",\n      \"journal\": \"ESC heart failure\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, RIP assay without full mechanistic reconstitution, limited follow-up on KIAA1522 itself\",\n      \"pmids\": [\"35730141\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NHSL3/KIAA1522 is a multi-isoform protein belonging to the Nance-Horan Syndrome family that regulates cell migration through at least two distinct isoform-specific mechanisms: a long isoform interacts with MENA/VASP proteins at cell-cell junctions to enable collective migration, while a short isoform interacts with 14-3-3θ in lamellipodia to modulate single-cell migration persistence; NHSL3 also co-localizes with and inhibits Scar/WAVE-Arp2/3 activity at lamellipodia by binding both the Abi and CYFIP1/2 subunits of the Scar/WAVE complex via short linear motifs; additionally, KIAA1522 promotes tumor cell proliferation and survival through activation of ERK, NFκB, Wnt/β-catenin, and Notch signaling pathways, and its expression is transcriptionally repressed by KLF9 and regulated epigenetically via the PLACT1-EHMT2-KLF2 axis, while isoform-specific nuclear functions regulate LTR retrotransposon activity in distinct pluripotency states.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NHSL3 (KIAA1522) is a multi-isoform member of the Nance-Horan Syndrome family that regulates actin-based cell migration through isoform-specific protein interactions at the leading edge and at cell-cell junctions [#0]. Loss of NHSL3 increases persistence of single-cell migration while impairing collective migration, reflecting two genetically separable functions [#0]: a long isoform binds MENA/VASP proteins at cell-cell junctions to enable collective migration, whereas a short isoform binds 14-3-3θ in lamellipodia to set single-cell migration persistence, with binding-site mutations abolishing each rescue [#1, #2]. At the lamellipodial edge NHSL3 co-localizes with Ena/VASP and the Scar/WAVE complex and inhibits Scar/WAVE-Arp2/3 activity [#3], engaging the complex through both its Abi and CYFIP1/2 subunits via three short linear motifs [#4]. Beyond motility, KIAA1522 promotes tumor cell proliferation, survival, and chemoresistance by activating ERK [#5], TNFα-TNFR2-NFκB [#6], Wnt/β-catenin [#7], and Notch [#8] signaling. Its expression is controlled transcriptionally by KLF9, which binds the KIAA1522 promoter and represses it [#9], and epigenetically through a PLACT1-EHMT2-KLF2 axis that de-represses KIAA1522 [#10]; alternative first-exon usage generates state-specific isoforms in pluripotent cells that differ in N-terminal domain, localization, and effect on LTR retrotransposon activity [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the first molecular handle on KIAA1522 function by linking it to a defined growth-signaling readout, addressing why its expression correlates with tumor aggressiveness.\",\n      \"evidence\": \"siRNA knockdown with phospho-ERK Western blot, proliferation/anoikis assays, and xenograft/metastasis models in esophageal squamous cell carcinoma\",\n      \"pmids\": [\"28794639\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not show direct molecular interaction between KIAA1522 and ERK pathway components\", \"Mechanism of ERK activation unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended KIAA1522's pro-tumor role to additional survival pathways, showing it potentiates NFκB and Wnt/β-catenin signaling and drives chemoresistance.\",\n      \"evidence\": \"In vivo KrasG12D/Cas9 model with AAV-sgRNA depletion plus NFκB inhibitor rescue (lung adenocarcinoma); TOP/FOP-flash luciferase reporters (hepatocellular carcinoma)\",\n      \"pmids\": [\"32854746\", \"32606779\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding partners connecting KIAA1522 to NFκB/Wnt not identified\", \"Whether pathway activation is direct or secondary unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Added Notch as a fourth oncogenic pathway downstream of KIAA1522, reinforcing its role in proliferation, invasion, and metastasis.\",\n      \"evidence\": \"Transcriptome sequencing, GSEA, Western blot, IHC and tail-vein metastasis model in colorectal cancer\",\n      \"pmids\": [\"34826587\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking KIAA1522 to Notch activation not defined\", \"No physical interaction demonstrated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Answered how KIAA1522 levels are set transcriptionally, identifying KLF9 as a direct promoter-binding repressor.\",\n      \"evidence\": \"ChIP and dual-luciferase reporter with overexpression/knockdown rescue in pancreatic cancer\",\n      \"pmids\": [\"38520660\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream regulators of KLF9 in this context not defined\", \"Single tumor type\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the core cell-biological function of NHSL3, demonstrating isoform-specific interactions that partition single-cell versus collective migration control.\",\n      \"evidence\": \"Knockout cell lines, wound healing and single-cell tracking, isoform-specific proteomics, AlphaFold2 binding prediction, and binding-site mutagenesis rescue (Nature Communications)\",\n      \"pmids\": [\"39747206\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the MENA/VASP and 14-3-3θ interactions not determined\", \"How isoform choice is regulated across tissues unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a molecular mechanism for NHSL3's effect on the actin machinery by showing it binds and inhibits the Scar/WAVE-Arp2/3 system.\",\n      \"evidence\": \"Co-localization imaging, pulldown/Co-IP, short linear motif mapping, and cellular Arp2/3 activity assay (bioRxiv preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.03.647056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"In vitro reconstitution of Arp2/3 inhibition not shown\", \"Functional consequence of dual Abi/CYFIP binding unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Uncovered an epigenetic axis and a nuclear, isoform-dependent function, broadening KIAA1522 beyond cytoplasmic migration into transcriptional/chromatin contexts.\",\n      \"evidence\": \"ChIP/RNA pulldown/xenograft for the PLACT1-EHMT2-KLF2 axis; RNA-seq, CUT&Tag and localization for state-specific ESC isoforms regulating LTR retrotransposons\",\n      \"pmids\": [\"41164921\", \"40553414\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct chromatin-binding activity of nuclear NHSL3 isoforms not characterized\", \"Mechanism by which isoforms affect LTR activity unknown\", \"Single lab per finding\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct isoform-specific cytoplasmic (migration), oncogenic signaling, and nuclear (retrotransposon) functions are coordinated within and across cell types remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying biochemical activity defined for the protein\", \"Relationship between migration role and oncogenic signaling not established\", \"No structural model of the protein\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"VASP\", \"ENAH\", \"YWHAQ\", \"ABI1\", \"CYFIP1\", \"CYFIP2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":4,"faith_total":4,"faith_pct":100.0}}