{"gene":"POTEE","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":2019,"finding":"POTEE drives colorectal cancer cell growth, cell-cycle progression, and inhibits apoptosis via a POTEE/SPHK1/p65 signaling axis, where POTEE overexpression increases SPHK1 protein expression and promotes phosphorylation/activation of p65.","method":"Microarray analysis, western blotting, xenograft tumor model, CRC cell knockdown/overexpression","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — single lab, multiple methods (microarray, western blot, xenograft) but pathway placement relies on correlative protein expression changes without direct enzymatic assays","pmids":["31723122"],"is_preprint":false},{"year":2018,"finding":"POTEE interacts with mTOR, RICTOR (mTORC2 components), and Rad51 in tumor-associated macrophages (TAMs), and siRNA-mediated knockdown of POTEE impairs cell survival of macrophages and TAMs, indicating POTEE activates mTORC2 signaling in these cells.","method":"Co-immunoprecipitation (protein-protein interaction), siRNA knockdown, immunofluorescence in macrophage cell models","journal":"Cellular immunology","confidence":"Medium","confidence_rationale":"Tier 3 + Moderate — single lab, protein-protein interaction by Co-IP with functional siRNA knockdown phenotype","pmids":["30420269"],"is_preprint":false},{"year":2018,"finding":"HIV-1 Nef interacts directly with POTEE (identified by pulldown and MALDI-TOF, validated by mammalian two-hybrid assay), and this Nef-POTEE interaction activates the mTORC2 complex (with mTOR and Rictor), leading to AKT and PKC-α activation and increased macrophage invasion and migration.","method":"Pull-down assay, MALDI-TOF, mammalian two-hybrid assay, co-immunoprecipitation, cell invasion and migration assays","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal interaction validated by multiple methods (pulldown, two-hybrid, Co-IP) with functional phenotype, single lab","pmids":["30391463"],"is_preprint":false},{"year":2020,"finding":"POTEE promotes colorectal carcinoma cell migration, invasion, and epithelial-mesenchymal transition (EMT) by activating the small GTPases Rac1 and Cdc42; POTEE was localized to the cytoplasm.","method":"qRT-PCR, western blotting, immunohistochemistry, siRNA knockdown/overexpression, in vitro migration/invasion assays, in vivo tumor metastasis model, Rac1/Cdc42 activation assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple methods including in vivo model and GTPase activation assays, single lab","pmids":["32142855"],"is_preprint":false},{"year":2020,"finding":"POTEE knockdown or GSK-3β inhibition attenuates proliferation of pancreatic cancer cells; POTEE stimulates proliferation via activation of the PI3K/Akt/GSK-3β/β-catenin signaling pathway, with downstream protein levels reduced upon POTEE knockdown.","method":"siRNA knockdown, GSK-3β inhibitor treatment (Tideglusib), western blotting for pathway components, proliferation assays","journal":"BioFactors (Oxford, England)","confidence":"Low","confidence_rationale":"Tier 3 + Weak — single lab, pathway inferred from protein level changes after knockdown without direct biochemical reconstitution","pmids":["32589786"],"is_preprint":false},{"year":2021,"finding":"N-myristoylation of target proteins (NDP and NUP groups) by NMT1 is POTEE-dependent; POTEE is required for NMT1-mediated N-myristoylation activity in liver cancer cells, affecting protein stability through differential ubiquitination by HIST1H4H E3 ligase.","method":"Click chemistry N-myristoylation assay, iTraq proteomics, conditional NMT1 knockout mouse model, parallel reaction monitoring (PRM)","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 — click chemistry assay directly measuring myristoylation with in vivo KO model, but POTEE-dependence mechanistic detail is from single lab","pmids":["34136404"],"is_preprint":false},{"year":2023,"finding":"POTEE is a novel effector of SUMOylated Rac1 (Rac1-SUMO1) in breast cancer; POTEE activates Rac1 at the invadopodium by recruiting the GEF TRIO, thereby driving invadopodium formation, tumor cell proliferation, and metastasis in vitro and in vivo.","method":"Co-immunoprecipitation, co-localization studies, invadopodium formation assays, in vitro/in vivo proliferation and metastasis assays, TRIO-GEF recruitment assay","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2-3 — mechanistic pathway placement (POTEE → TRIO recruitment → Rac1-SUMO1 activation → invadopodia) with in vitro and in vivo validation, single lab","pmids":["38098337"],"is_preprint":false},{"year":2025,"finding":"LINC00667 lncRNA binds directly to POTEE protein (confirmed by CHIRP and RIP assays) and promotes TRIM33-mediated ubiquitination and proteasomal degradation of POTEE; POTEE degradation reduces mitochondrial oxidative phosphorylation (OXPHOS) complex expression, suppressing breast cancer progression.","method":"CHIRP assay, RIP assay, cycloheximide chase, MG132 proteasome inhibitor treatment, siRNA knockdown, overexpression, OXPHOS complex western blotting","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 — direct RNA-protein interaction validated by orthogonal methods (CHIRP + RIP), CHX chase establishing degradation kinetics, TRIM33 identified as E3 ligase, single lab","pmids":["40834976"],"is_preprint":false},{"year":2024,"finding":"MARK1 kinase directly binds POTEE (validated by luciferase reporter assay) and negatively regulates POTEE expression; MARK1 overexpression suppresses POTEE and inhibits sorafenib-resistant HCC cell proliferation, an effect reversed by co-overexpression of POTEE.","method":"Luciferase reporter binding assay, qRT-PCR, rescue/epistasis experiment (MARK1 + POTEE co-overexpression), sorafenib-resistant cell model","journal":"Open medicine (Warsaw, Poland)","confidence":"Low","confidence_rationale":"Tier 3 + Weak — single lab, luciferase reporter for binding and epistasis rescue, no direct biochemical mechanism for how MARK1 suppresses POTEE","pmids":["39534429"],"is_preprint":false}],"current_model":"POTEE (POTE Ankyrin Domain Family Member E) is a cytoplasmic cancer-testis antigen that promotes tumor cell proliferation, migration, invasion, and metastasis through multiple signaling axes: it acts as an effector of SUMOylated Rac1 by recruiting the GEF TRIO to drive invadopodium formation; it activates Rac1/Cdc42 to promote EMT; it upregulates SPHK1 to activate NF-κB/p65; it activates PI3K/Akt/GSK-3β/β-catenin signaling; it facilitates mTORC2 activation (with mTOR and RICTOR) in macrophages; and its stability is regulated by TRIM33-mediated ubiquitination downstream of the lncRNA LINC00667, while MARK1 negatively regulates its expression in hepatocellular carcinoma."},"narrative":{"teleology":[{"year":2018,"claim":"The discovery that POTEE physically interacts with mTORC2 components (mTOR, RICTOR) and that its knockdown impairs macrophage survival established POTEE as a signaling-active protein rather than merely a cancer-testis marker, linking it to a defined kinase signaling complex.","evidence":"Co-immunoprecipitation and siRNA knockdown in macrophage and tumor-associated macrophage cell models","pmids":["30420269","30391463"],"confidence":"Medium","gaps":["No direct kinase assay showing POTEE stimulates mTORC2 catalytic activity","Mechanism by which POTEE promotes mTORC2 assembly or activation is undefined","Findings limited to macrophage lineage; generalizability uncertain"]},{"year":2019,"claim":"Identification of the POTEE/SPHK1/p65 axis in colorectal cancer demonstrated that POTEE acts upstream of NF-κB signaling by increasing SPHK1 protein levels, providing the first cancer-intrinsic signaling pathway downstream of POTEE.","evidence":"Microarray, western blotting, overexpression/knockdown in CRC cells, and xenograft tumor model","pmids":["31723122"],"confidence":"Medium","gaps":["No direct biochemical mechanism for how POTEE upregulates SPHK1","Pathway ordering inferred from correlative protein level changes","Single lab finding without independent replication"]},{"year":2020,"claim":"Demonstration that POTEE activates Rac1 and Cdc42 GTPases to drive EMT, migration, and invasion established small GTPase activation as a core effector mechanism of POTEE in colorectal cancer, while a parallel study linked POTEE to PI3K/Akt/GSK-3β/β-catenin in pancreatic cancer proliferation.","evidence":"GTPase activation pull-down assays, siRNA knockdown/overexpression, in vivo metastasis model (CRC); siRNA knockdown with GSK-3β inhibitor epistasis and western blotting (pancreatic cancer)","pmids":["32142855","32589786"],"confidence":"Medium","gaps":["PI3K/Akt pathway activation inferred from protein level changes without direct biochemical reconstitution (Low confidence for pancreatic cancer study)","Whether Rac1/Cdc42 and PI3K/Akt represent parallel or converging mechanisms is unknown","No GEF or GAP identified mediating POTEE-dependent GTPase activation at this stage"]},{"year":2021,"claim":"Discovery that POTEE is required for NMT1-mediated N-myristoylation of specific substrate proteins expanded POTEE's role beyond signaling scaffold to a co-factor for a lipid modification enzyme, connecting it to protein stability through differential ubiquitination.","evidence":"Click chemistry myristoylation assay, iTRAQ proteomics, conditional NMT1 knockout mouse model, PRM in liver cancer cells","pmids":["34136404"],"confidence":"Medium","gaps":["Molecular mechanism by which POTEE enables NMT1 activity is not defined","Whether POTEE is a direct NMT1 binding partner or acts indirectly is unresolved","Relationship of NMT1/myristoylation function to POTEE's GTPase-activating roles is unclear"]},{"year":2023,"claim":"Identification of POTEE as an effector of SUMOylated Rac1 that recruits the GEF TRIO to invadopodia resolved the upstream signal (Rac1-SUMO1) and the direct mechanism (TRIO recruitment) by which POTEE activates Rac1 at sites of invasion, unifying earlier GTPase activation findings with a defined molecular cascade.","evidence":"Co-immunoprecipitation, co-localization, TRIO-GEF recruitment assays, invadopodium formation assays, and in vivo metastasis assays in breast cancer models","pmids":["38098337"],"confidence":"Medium","gaps":["Structural basis of POTEE-TRIO and POTEE-SUMOylated Rac1 interactions is unknown","Whether TRIO recruitment mechanism operates in cancer types beyond breast cancer is untested","Single lab; no independent confirmation of the SUMOylated Rac1–POTEE–TRIO cascade"]},{"year":2024,"claim":"Finding that MARK1 kinase binds POTEE and negatively regulates its expression identified the first upstream kinase-level negative regulator of POTEE, relevant to sorafenib resistance in hepatocellular carcinoma.","evidence":"Luciferase reporter binding assay, rescue/epistasis experiment with co-overexpression, sorafenib-resistant HCC cell model","pmids":["39534429"],"confidence":"Low","gaps":["No direct biochemical mechanism for how MARK1 suppresses POTEE expression (phosphorylation site, degradation, transcriptional effect all undefined)","Luciferase reporter binding assay is non-standard for protein-protein interaction validation","Single lab, single cancer model"]},{"year":2025,"claim":"Demonstration that the lncRNA LINC00667 directly binds POTEE protein and promotes TRIM33-mediated ubiquitination and proteasomal degradation revealed the first defined post-translational regulatory mechanism controlling POTEE protein levels, and linked POTEE stability to mitochondrial OXPHOS complex expression.","evidence":"CHIRP and RIP assays for RNA-protein interaction, cycloheximide chase and MG132 treatment, OXPHOS complex western blotting in breast cancer cells","pmids":["40834976"],"confidence":"Medium","gaps":["TRIM33 ubiquitination sites on POTEE are not mapped","How POTEE maintains OXPHOS complex expression is mechanistically undefined","Whether LINC00667/TRIM33 axis regulates POTEE in non-breast tissues is unknown"]},{"year":null,"claim":"The structural basis of POTEE's multi-pathway scaffolding activity — how a single protein engages TRIO, mTORC2 components, and NMT1, and whether these represent mutually exclusive or concurrent interactions — remains undefined, and no crystal or cryo-EM structure of POTEE or its complexes has been reported.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of POTEE or any of its complexes exists","Domain-interaction mapping for individual binding partners is incomplete","All pathway studies originate from single labs without independent replication"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,4,6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,3,4,6]}],"complexes":[],"partners":["TRIO","MTOR","RICTOR","SPHK1","NMT1","TRIM33","MARK1"],"other_free_text":[]},"mechanistic_narrative":"POTEE is a cancer-testis antigen that functions as a pro-tumorigenic signaling scaffold, promoting cell proliferation, migration, invasion, and metastasis across multiple cancer types by activating Rac1/Cdc42 small GTPases and downstream oncogenic pathways. POTEE serves as an effector of SUMOylated Rac1, recruiting the guanine nucleotide exchange factor TRIO to drive invadopodium formation in breast cancer [PMID:38098337], and independently activates Rac1/Cdc42 to promote epithelial-mesenchymal transition in colorectal carcinoma [PMID:32142855]. POTEE also engages the mTORC2 complex through interactions with mTOR and RICTOR in macrophages, activating AKT and PKC-α signaling [PMID:30420269, PMID:30391463], and stimulates PI3K/Akt/GSK-3β/β-catenin and SPHK1/NF-κB/p65 axes to drive proliferation in pancreatic and colorectal cancers [PMID:32589786, PMID:31723122]. POTEE protein stability is regulated by TRIM33-mediated ubiquitination, which is facilitated by direct binding of the lncRNA LINC00667, and POTEE degradation reduces mitochondrial OXPHOS complex expression [PMID:40834976]."},"prefetch_data":{"uniprot":{"accession":"Q6S8J3","full_name":"POTE ankyrin domain family member E","aliases":["ANKRD26-like family C member 1A","Prostate, ovary, testis-expressed protein on chromosome 2","POTE-2"],"length_aa":1075,"mass_kda":121.4,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q6S8J3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/POTEE","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1047,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ACTB","stoichiometry":0.2},{"gene":"CALD1","stoichiometry":0.2},{"gene":"DEGS1","stoichiometry":0.2},{"gene":"FKBP5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/POTEE","total_profiled":1310},"omim":[{"mim_id":"612863","title":"CHROMOSOME 6q24-q25 DELETION SYNDROME","url":"https://www.omim.org/entry/612863"},{"mim_id":"608914","title":"POTE ANKYRIN DOMAIN FAMILY, MEMBER E; POTEE","url":"https://www.omim.org/entry/608914"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Not detected","tissue_distribution":"Not detected","driving_tissues":[],"url":"https://www.proteinatlas.org/search/POTEE"},"hgnc":{"alias_symbol":["POTE2","POTE-2","A26C1","POTE2gamma","CT104.2"],"prev_symbol":["A26C1A"]},"alphafold":{"accession":"Q6S8J3","domains":[{"cath_id":"1.25.40.20","chopping":"127-367","consensus_level":"medium","plddt":91.0483,"start":127,"end":367},{"cath_id":"3.30.420.40","chopping":"709-741_751-846_1042-1075","consensus_level":"medium","plddt":93.5849,"start":709,"end":1075}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6S8J3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6S8J3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6S8J3-F1-predicted_aligned_error_v6.png","plddt_mean":70.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=POTEE","jax_strain_url":"https://www.jax.org/strain/search?query=POTEE"},"sequence":{"accession":"Q6S8J3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6S8J3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6S8J3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6S8J3"}},"corpus_meta":[{"pmid":"24969553","id":"PMC_24969553","title":"Identification of ApoA1, HPX and POTEE genes by omic analysis in breast cancer.","date":"2014","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/24969553","citation_count":37,"is_preprint":false},{"pmid":"31723122","id":"PMC_31723122","title":"POTEE drives colorectal cancer development via regulating SPHK1/p65 signaling.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/31723122","citation_count":29,"is_preprint":false},{"pmid":"25860145","id":"PMC_25860145","title":"Serum levels of the cancer-testis antigen POTEE and its clinical significance in non-small-cell lung cancer.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25860145","citation_count":14,"is_preprint":false},{"pmid":"34136404","id":"PMC_34136404","title":"N-Myristoylation by NMT1 Is POTEE-Dependent to Stimulate Liver Tumorigenesis via Differentially Regulating Ubiquitination of Targets.","date":"2021","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/34136404","citation_count":11,"is_preprint":false},{"pmid":"30420269","id":"PMC_30420269","title":"Identification of MΦ specific POTEE expression: Its role in mTORC2 activation via protein-protein interaction in TAMs.","date":"2018","source":"Cellular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30420269","citation_count":11,"is_preprint":false},{"pmid":"30391463","id":"PMC_30391463","title":"HIV-1 Nef-POTEE; A novel interaction modulates macrophage dissemination via mTORC2 signaling pathway.","date":"2018","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/30391463","citation_count":9,"is_preprint":false},{"pmid":"32589786","id":"PMC_32589786","title":"POTEE stimulates the proliferation of pancreatic cancer by activating the PI3K/Akt/GSK-3β/β-catenin signaling.","date":"2020","source":"BioFactors (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/32589786","citation_count":9,"is_preprint":false},{"pmid":"32142855","id":"PMC_32142855","title":"POTEE promotes colorectal carcinoma progression via activating the Rac1/Cdc42 pathway.","date":"2020","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/32142855","citation_count":8,"is_preprint":false},{"pmid":"38098337","id":"PMC_38098337","title":"POTEE promotes breast cancer cell malignancy by inducing invadopodia formation through the activation of SUMOylated Rac1.","date":"2023","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38098337","citation_count":4,"is_preprint":false},{"pmid":"39891409","id":"PMC_39891409","title":"Identification of TTLL8, POTEE, and PKMYT1 as immunogenic cancer-associated antigens and potential immunotherapy targets in ovarian cancer.","date":"2025","source":"Oncoimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/39891409","citation_count":2,"is_preprint":false},{"pmid":"37318657","id":"PMC_37318657","title":"POTEE mutation as a potential predictive biomarker for immune checkpoint inhibitors in lung adenocarcinoma.","date":"2023","source":"Investigational new drugs","url":"https://pubmed.ncbi.nlm.nih.gov/37318657","citation_count":2,"is_preprint":false},{"pmid":"40834976","id":"PMC_40834976","title":"LncRNA LINC00667 inhibits breast cancer progression by regulating POTEE to suppress mitochondrial oxidative phosphorylation.","date":"2025","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/40834976","citation_count":1,"is_preprint":false},{"pmid":"39534429","id":"PMC_39534429","title":"MARK1 suppress malignant progression of hepatocellular carcinoma and improves sorafenib resistance through negatively regulating POTEE.","date":"2024","source":"Open medicine (Warsaw, Poland)","url":"https://pubmed.ncbi.nlm.nih.gov/39534429","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7731,"output_tokens":2327,"usd":0.029049},"stage2":{"model":"claude-opus-4-6","input_tokens":5661,"output_tokens":2470,"usd":0.135082},"total_usd":0.164131,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"POTEE drives colorectal cancer cell growth, cell-cycle progression, and inhibits apoptosis via a POTEE/SPHK1/p65 signaling axis, where POTEE overexpression increases SPHK1 protein expression and promotes phosphorylation/activation of p65.\",\n      \"method\": \"Microarray analysis, western blotting, xenograft tumor model, CRC cell knockdown/overexpression\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — single lab, multiple methods (microarray, western blot, xenograft) but pathway placement relies on correlative protein expression changes without direct enzymatic assays\",\n      \"pmids\": [\"31723122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"POTEE interacts with mTOR, RICTOR (mTORC2 components), and Rad51 in tumor-associated macrophages (TAMs), and siRNA-mediated knockdown of POTEE impairs cell survival of macrophages and TAMs, indicating POTEE activates mTORC2 signaling in these cells.\",\n      \"method\": \"Co-immunoprecipitation (protein-protein interaction), siRNA knockdown, immunofluorescence in macrophage cell models\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 + Moderate — single lab, protein-protein interaction by Co-IP with functional siRNA knockdown phenotype\",\n      \"pmids\": [\"30420269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HIV-1 Nef interacts directly with POTEE (identified by pulldown and MALDI-TOF, validated by mammalian two-hybrid assay), and this Nef-POTEE interaction activates the mTORC2 complex (with mTOR and Rictor), leading to AKT and PKC-α activation and increased macrophage invasion and migration.\",\n      \"method\": \"Pull-down assay, MALDI-TOF, mammalian two-hybrid assay, co-immunoprecipitation, cell invasion and migration assays\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal interaction validated by multiple methods (pulldown, two-hybrid, Co-IP) with functional phenotype, single lab\",\n      \"pmids\": [\"30391463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"POTEE promotes colorectal carcinoma cell migration, invasion, and epithelial-mesenchymal transition (EMT) by activating the small GTPases Rac1 and Cdc42; POTEE was localized to the cytoplasm.\",\n      \"method\": \"qRT-PCR, western blotting, immunohistochemistry, siRNA knockdown/overexpression, in vitro migration/invasion assays, in vivo tumor metastasis model, Rac1/Cdc42 activation assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple methods including in vivo model and GTPase activation assays, single lab\",\n      \"pmids\": [\"32142855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"POTEE knockdown or GSK-3β inhibition attenuates proliferation of pancreatic cancer cells; POTEE stimulates proliferation via activation of the PI3K/Akt/GSK-3β/β-catenin signaling pathway, with downstream protein levels reduced upon POTEE knockdown.\",\n      \"method\": \"siRNA knockdown, GSK-3β inhibitor treatment (Tideglusib), western blotting for pathway components, proliferation assays\",\n      \"journal\": \"BioFactors (Oxford, England)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 + Weak — single lab, pathway inferred from protein level changes after knockdown without direct biochemical reconstitution\",\n      \"pmids\": [\"32589786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"N-myristoylation of target proteins (NDP and NUP groups) by NMT1 is POTEE-dependent; POTEE is required for NMT1-mediated N-myristoylation activity in liver cancer cells, affecting protein stability through differential ubiquitination by HIST1H4H E3 ligase.\",\n      \"method\": \"Click chemistry N-myristoylation assay, iTraq proteomics, conditional NMT1 knockout mouse model, parallel reaction monitoring (PRM)\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — click chemistry assay directly measuring myristoylation with in vivo KO model, but POTEE-dependence mechanistic detail is from single lab\",\n      \"pmids\": [\"34136404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"POTEE is a novel effector of SUMOylated Rac1 (Rac1-SUMO1) in breast cancer; POTEE activates Rac1 at the invadopodium by recruiting the GEF TRIO, thereby driving invadopodium formation, tumor cell proliferation, and metastasis in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, co-localization studies, invadopodium formation assays, in vitro/in vivo proliferation and metastasis assays, TRIO-GEF recruitment assay\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — mechanistic pathway placement (POTEE → TRIO recruitment → Rac1-SUMO1 activation → invadopodia) with in vitro and in vivo validation, single lab\",\n      \"pmids\": [\"38098337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LINC00667 lncRNA binds directly to POTEE protein (confirmed by CHIRP and RIP assays) and promotes TRIM33-mediated ubiquitination and proteasomal degradation of POTEE; POTEE degradation reduces mitochondrial oxidative phosphorylation (OXPHOS) complex expression, suppressing breast cancer progression.\",\n      \"method\": \"CHIRP assay, RIP assay, cycloheximide chase, MG132 proteasome inhibitor treatment, siRNA knockdown, overexpression, OXPHOS complex western blotting\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct RNA-protein interaction validated by orthogonal methods (CHIRP + RIP), CHX chase establishing degradation kinetics, TRIM33 identified as E3 ligase, single lab\",\n      \"pmids\": [\"40834976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARK1 kinase directly binds POTEE (validated by luciferase reporter assay) and negatively regulates POTEE expression; MARK1 overexpression suppresses POTEE and inhibits sorafenib-resistant HCC cell proliferation, an effect reversed by co-overexpression of POTEE.\",\n      \"method\": \"Luciferase reporter binding assay, qRT-PCR, rescue/epistasis experiment (MARK1 + POTEE co-overexpression), sorafenib-resistant cell model\",\n      \"journal\": \"Open medicine (Warsaw, Poland)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 + Weak — single lab, luciferase reporter for binding and epistasis rescue, no direct biochemical mechanism for how MARK1 suppresses POTEE\",\n      \"pmids\": [\"39534429\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"POTEE (POTE Ankyrin Domain Family Member E) is a cytoplasmic cancer-testis antigen that promotes tumor cell proliferation, migration, invasion, and metastasis through multiple signaling axes: it acts as an effector of SUMOylated Rac1 by recruiting the GEF TRIO to drive invadopodium formation; it activates Rac1/Cdc42 to promote EMT; it upregulates SPHK1 to activate NF-κB/p65; it activates PI3K/Akt/GSK-3β/β-catenin signaling; it facilitates mTORC2 activation (with mTOR and RICTOR) in macrophages; and its stability is regulated by TRIM33-mediated ubiquitination downstream of the lncRNA LINC00667, while MARK1 negatively regulates its expression in hepatocellular carcinoma.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"POTEE is a cancer-testis antigen that functions as a pro-tumorigenic signaling scaffold, promoting cell proliferation, migration, invasion, and metastasis across multiple cancer types by activating Rac1/Cdc42 small GTPases and downstream oncogenic pathways. POTEE serves as an effector of SUMOylated Rac1, recruiting the guanine nucleotide exchange factor TRIO to drive invadopodium formation in breast cancer [PMID:38098337], and independently activates Rac1/Cdc42 to promote epithelial-mesenchymal transition in colorectal carcinoma [PMID:32142855]. POTEE also engages the mTORC2 complex through interactions with mTOR and RICTOR in macrophages, activating AKT and PKC-α signaling [PMID:30420269, PMID:30391463], and stimulates PI3K/Akt/GSK-3β/β-catenin and SPHK1/NF-κB/p65 axes to drive proliferation in pancreatic and colorectal cancers [PMID:32589786, PMID:31723122]. POTEE protein stability is regulated by TRIM33-mediated ubiquitination, which is facilitated by direct binding of the lncRNA LINC00667, and POTEE degradation reduces mitochondrial OXPHOS complex expression [PMID:40834976].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"The discovery that POTEE physically interacts with mTORC2 components (mTOR, RICTOR) and that its knockdown impairs macrophage survival established POTEE as a signaling-active protein rather than merely a cancer-testis marker, linking it to a defined kinase signaling complex.\",\n      \"evidence\": \"Co-immunoprecipitation and siRNA knockdown in macrophage and tumor-associated macrophage cell models\",\n      \"pmids\": [\"30420269\", \"30391463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct kinase assay showing POTEE stimulates mTORC2 catalytic activity\",\n        \"Mechanism by which POTEE promotes mTORC2 assembly or activation is undefined\",\n        \"Findings limited to macrophage lineage; generalizability uncertain\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of the POTEE/SPHK1/p65 axis in colorectal cancer demonstrated that POTEE acts upstream of NF-κB signaling by increasing SPHK1 protein levels, providing the first cancer-intrinsic signaling pathway downstream of POTEE.\",\n      \"evidence\": \"Microarray, western blotting, overexpression/knockdown in CRC cells, and xenograft tumor model\",\n      \"pmids\": [\"31723122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct biochemical mechanism for how POTEE upregulates SPHK1\",\n        \"Pathway ordering inferred from correlative protein level changes\",\n        \"Single lab finding without independent replication\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that POTEE activates Rac1 and Cdc42 GTPases to drive EMT, migration, and invasion established small GTPase activation as a core effector mechanism of POTEE in colorectal cancer, while a parallel study linked POTEE to PI3K/Akt/GSK-3β/β-catenin in pancreatic cancer proliferation.\",\n      \"evidence\": \"GTPase activation pull-down assays, siRNA knockdown/overexpression, in vivo metastasis model (CRC); siRNA knockdown with GSK-3β inhibitor epistasis and western blotting (pancreatic cancer)\",\n      \"pmids\": [\"32142855\", \"32589786\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"PI3K/Akt pathway activation inferred from protein level changes without direct biochemical reconstitution (Low confidence for pancreatic cancer study)\",\n        \"Whether Rac1/Cdc42 and PI3K/Akt represent parallel or converging mechanisms is unknown\",\n        \"No GEF or GAP identified mediating POTEE-dependent GTPase activation at this stage\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that POTEE is required for NMT1-mediated N-myristoylation of specific substrate proteins expanded POTEE's role beyond signaling scaffold to a co-factor for a lipid modification enzyme, connecting it to protein stability through differential ubiquitination.\",\n      \"evidence\": \"Click chemistry myristoylation assay, iTRAQ proteomics, conditional NMT1 knockout mouse model, PRM in liver cancer cells\",\n      \"pmids\": [\"34136404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular mechanism by which POTEE enables NMT1 activity is not defined\",\n        \"Whether POTEE is a direct NMT1 binding partner or acts indirectly is unresolved\",\n        \"Relationship of NMT1/myristoylation function to POTEE's GTPase-activating roles is unclear\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of POTEE as an effector of SUMOylated Rac1 that recruits the GEF TRIO to invadopodia resolved the upstream signal (Rac1-SUMO1) and the direct mechanism (TRIO recruitment) by which POTEE activates Rac1 at sites of invasion, unifying earlier GTPase activation findings with a defined molecular cascade.\",\n      \"evidence\": \"Co-immunoprecipitation, co-localization, TRIO-GEF recruitment assays, invadopodium formation assays, and in vivo metastasis assays in breast cancer models\",\n      \"pmids\": [\"38098337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis of POTEE-TRIO and POTEE-SUMOylated Rac1 interactions is unknown\",\n        \"Whether TRIO recruitment mechanism operates in cancer types beyond breast cancer is untested\",\n        \"Single lab; no independent confirmation of the SUMOylated Rac1–POTEE–TRIO cascade\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Finding that MARK1 kinase binds POTEE and negatively regulates its expression identified the first upstream kinase-level negative regulator of POTEE, relevant to sorafenib resistance in hepatocellular carcinoma.\",\n      \"evidence\": \"Luciferase reporter binding assay, rescue/epistasis experiment with co-overexpression, sorafenib-resistant HCC cell model\",\n      \"pmids\": [\"39534429\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct biochemical mechanism for how MARK1 suppresses POTEE expression (phosphorylation site, degradation, transcriptional effect all undefined)\",\n        \"Luciferase reporter binding assay is non-standard for protein-protein interaction validation\",\n        \"Single lab, single cancer model\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstration that the lncRNA LINC00667 directly binds POTEE protein and promotes TRIM33-mediated ubiquitination and proteasomal degradation revealed the first defined post-translational regulatory mechanism controlling POTEE protein levels, and linked POTEE stability to mitochondrial OXPHOS complex expression.\",\n      \"evidence\": \"CHIRP and RIP assays for RNA-protein interaction, cycloheximide chase and MG132 treatment, OXPHOS complex western blotting in breast cancer cells\",\n      \"pmids\": [\"40834976\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"TRIM33 ubiquitination sites on POTEE are not mapped\",\n        \"How POTEE maintains OXPHOS complex expression is mechanistically undefined\",\n        \"Whether LINC00667/TRIM33 axis regulates POTEE in non-breast tissues is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of POTEE's multi-pathway scaffolding activity — how a single protein engages TRIO, mTORC2 components, and NMT1, and whether these represent mutually exclusive or concurrent interactions — remains undefined, and no crystal or cryo-EM structure of POTEE or its complexes has been reported.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural model of POTEE or any of its complexes exists\",\n        \"Domain-interaction mapping for individual binding partners is incomplete\",\n        \"All pathway studies originate from single labs without independent replication\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 3, 4, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TRIO\", \"mTOR\", \"RICTOR\", \"SPHK1\", \"NMT1\", \"TRIM33\", \"MARK1\"],\n    \"other_free_text\": []\n  }\n}\n```"}