{"gene":"POTEE","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2018,"finding":"POTEE interacts with mTOR, RICTOR, and Rad51 in tumor-associated macrophages (TAMs), and this protein-protein interaction activates mTORC2, promoting cell invasion. siRNA-mediated knockdown of POTEE impairs cell survival in macrophages and TAMs.","method":"Co-immunoprecipitation (protein-protein interaction), siRNA knockdown with cell survival and invasion assays","journal":"Cellular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional siRNA knockdown in a single lab with multiple orthogonal assays","pmids":["30420269"],"is_preprint":false},{"year":2018,"finding":"HIV-1 Nef directly interacts with POTEE (identified by pull-down and MALDI-TOF, validated by mammalian two-hybrid assay). The Nef-POTEE interaction recruits mTOR and RICTOR to form/activate mTORC2, which in turn activates AKT and PKC-α, leading to increased macrophage invasion and migration.","method":"Pull-down assay, MALDI-TOF mass spectrometry, mammalian two-hybrid assay, immunoprecipitation, cell invasion/migration assays","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pull-down, two-hybrid, Co-IP, functional assays) in a single lab","pmids":["30391463"],"is_preprint":false},{"year":2019,"finding":"POTEE promotes colorectal cancer cell growth, cell-cycle progression, and inhibits apoptosis via a POTEE/SPHK1/p65 signaling axis: overexpression of POTEE increases SPHK1 protein levels, which promotes phosphorylation and activation of p65.","method":"Microarray analysis, western blotting, overexpression and knockdown experiments, xenograft tumor model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional loss/gain-of-function with pathway validation by western blot, single lab","pmids":["31723122"],"is_preprint":false},{"year":2020,"finding":"POTEE is localized in the cytoplasm of colorectal carcinoma cells and promotes CRC migration, invasion, and epithelial-mesenchymal transition (EMT) by increasing activation of the small GTPases Rac1 and Cdc42.","method":"Immunohistochemistry (subcellular localization), qRT-PCR, western blotting, knockdown/overexpression, in vitro migration/invasion assays, in vivo xenograft, Rac1/Cdc42 activity assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional KD/OE with defined pathway readout (Rac1/Cdc42 activation), single lab, multiple orthogonal methods","pmids":["32142855"],"is_preprint":false},{"year":2020,"finding":"POTEE knockdown or GSK-3β inhibition attenuates proliferation of pancreatic cancer cells, and POTEE activates the PI3K/Akt/GSK-3β/β-catenin signaling pathway, as shown by downregulation of key pathway proteins upon POTEE knockdown.","method":"siRNA knockdown, GSK-3β inhibitor (Tideglusib), proliferation assays, western blotting of PI3K/Akt/GSK-3β/β-catenin pathway components","journal":"BioFactors (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional KD with pharmacological inhibitor rescue and pathway protein quantification, single lab","pmids":["32589786"],"is_preprint":false},{"year":2021,"finding":"N-myristoylation of POTEE targets (NDP and NUP protein categories) by NMT1 is dependent on POTEE; POTEE is required for NMT1-mediated N-myristoylation of these substrates. N-myristoylation differentially regulates protein stability: NDP proteins are destabilized (via RPL7A/HIST1H4H ubiquitin E3 ligase-mediated ubiquitination), while NUP proteins are stabilized (protected from ubiquitination by HBB).","method":"Click chemistry N-myristoylation assay, proteomics (iTRAQ), liver conditional NMT1 knockout mouse model, parallel reaction monitoring (PRM)","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — click chemistry enzymatic assay and in vivo KO model, single lab, multiple methods","pmids":["34136404"],"is_preprint":false},{"year":2023,"finding":"POTEE is a Rac1-SUMO1 effector: it co-localizes with SUMOylated Rac1 (Rac1-SUMO1) at invadopodia, recruits TRIO-GEF to activate Rac1 in the invadopodium, and thereby drives invadopodium formation, tumor cell proliferation, and metastasis in breast cancer cells in vitro and in vivo.","method":"Co-localization studies, invadopodium formation assays, TRIO-GEF recruitment assays, in vitro and in vivo tumor proliferation and metastasis assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assays with defined molecular interaction (Rac1-SUMO1/TRIO-GEF recruitment) in vitro and in vivo, single lab","pmids":["38098337"],"is_preprint":false},{"year":2024,"finding":"MARK1 directly binds to POTEE (confirmed by luciferase reporter assay) and negatively regulates POTEE expression. Overexpression of MARK1 reduces POTEE levels and suppresses cell proliferation in sorafenib-resistant hepatocellular carcinoma cells; co-overexpression of POTEE reverses the MARK1-mediated suppression.","method":"Luciferase reporter assay, qRT-PCR, overexpression/rescue experiments in sorafenib-resistant cell models","journal":"Open medicine (Warsaw, Poland)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, luciferase binding assay and overexpression rescue without biochemical interaction confirmation","pmids":["39534429"],"is_preprint":false},{"year":2025,"finding":"LINC00667 lncRNA directly binds POTEE protein (confirmed by CHIRP and RIP assays) and promotes TRIM33-mediated ubiquitination and proteasomal degradation of POTEE (demonstrated by cycloheximide chase and MG132 rescue). Degradation of POTEE decreases mitochondrial oxidative phosphorylation (OXPHOS) complex expression, while POTEE overexpression enhances OXPHOS activity.","method":"CHIRP assay, RIP assay, cycloheximide chase experiment, MG132 proteasome inhibitor treatment, OXPHOS complex expression by western blotting, knockdown/overexpression","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (CHIRP, RIP, CHX chase, proteasome inhibitor rescue) in a single lab identifying TRIM33 as E3 ligase and mitochondrial OXPHOS as functional output","pmids":["40834976"],"is_preprint":false}],"current_model":"POTEE is a cytoplasmic cancer-testis antigen that functions as an oncogenic scaffold/effector protein: it activates Rac1 (via recruitment of TRIO-GEF and interaction with SUMOylated Rac1) to drive invadopodium formation and metastasis; it signals through SPHK1/p65 and PI3K/Akt/GSK-3β/β-catenin axes to promote proliferation; it interacts with mTOR/RICTOR complexes (mTORC2) to activate AKT and PKC-α, enhancing macrophage and tumor cell invasion; it is required for NMT1-mediated N-myristoylation of specific substrates; it promotes mitochondrial OXPHOS and is subject to TRIM33-mediated ubiquitination/degradation regulated by LINC00667; and its expression is negatively regulated by MARK1 kinase through direct binding."},"narrative":{"mechanistic_narrative":"POTEE is a cytoplasmic oncogenic effector protein that promotes tumor cell proliferation, invasion, and metastasis across multiple cancer types by coupling to small-GTPase and growth-signaling pathways [PMID:32142855, PMID:38098337]. A central activity is activation of the Rho-family GTPases Rac1 and Cdc42: in colorectal carcinoma POTEE elevates Rac1/Cdc42 activity to drive migration, invasion, and epithelial-mesenchymal transition [PMID:32142855], and in breast cancer it co-localizes with SUMOylated Rac1 (Rac1-SUMO1) at invadopodia where it recruits the TRIO guanine-nucleotide exchange factor to locally activate Rac1, driving invadopodium formation and metastasis [PMID:38098337]. POTEE additionally engages mitogenic signaling through the SPHK1/p65 axis to promote cell-cycle progression and suppress apoptosis [PMID:31723122] and through the PI3K/Akt/GSK-3β/β-catenin pathway to support proliferation [PMID:32589786]. In macrophages it scaffolds mTOR and RICTOR to assemble and activate mTORC2, which activates AKT and PKC-α to enhance invasion, an interaction co-opted by HIV-1 Nef [PMID:30420269, PMID:30391463]. POTEE is also required for NMT1-mediated N-myristoylation of a defined set of substrate proteins [PMID:34136404] and sustains mitochondrial oxidative phosphorylation, with its protein levels controlled by TRIM33-mediated ubiquitin-proteasomal degradation directed by the lncRNA LINC00667 [PMID:40834976] and negatively regulated by MARK1 [PMID:39534429].","teleology":[{"year":2018,"claim":"Established that POTEE acts as a scaffold linking to growth/survival signaling by physically assembling the mTORC2 machinery, answering how POTEE could influence invasion at the molecular level.","evidence":"Co-immunoprecipitation of POTEE with mTOR/RICTOR/Rad51 plus siRNA knockdown with survival and invasion assays in macrophages and TAMs; independently, pull-down/MALDI-TOF/two-hybrid showing HIV-1 Nef bridges POTEE to mTOR/RICTOR to activate AKT and PKC-α","pmids":["30420269","30391463"],"confidence":"Medium","gaps":["Direct vs. indirect nature of the POTEE–mTOR/RICTOR contact not resolved","Mapping of interaction domains absent","Findings confined to macrophage/TAM context"]},{"year":2019,"claim":"Linked POTEE to a defined proliferative signaling axis, showing it drives growth and blocks apoptosis through SPHK1-dependent p65 activation.","evidence":"Microarray, gain/loss-of-function, western blotting, and xenograft tumor model in colorectal cancer","pmids":["31723122"],"confidence":"Medium","gaps":["Mechanism by which POTEE raises SPHK1 protein levels unknown","No direct biochemical interaction demonstrated"]},{"year":2020,"claim":"Defined POTEE's core motility function as activation of Rho-family GTPases and engagement of PI3K/Akt signaling, connecting it to invasion, EMT, and proliferation.","evidence":"Cytoplasmic localization by IHC, knockdown/overexpression with Rac1/Cdc42 activity assays and migration/invasion in CRC; siRNA knockdown plus GSK-3β inhibitor rescue and pathway western blotting in pancreatic cancer","pmids":["32142855","32589786"],"confidence":"Medium","gaps":["How POTEE activates Rac1/Cdc42 not yet mechanistically defined","Direct vs. indirect engagement of PI3K/Akt pathway unresolved"]},{"year":2021,"claim":"Revealed a role for POTEE in protein lipidation, showing it is required for NMT1-mediated N-myristoylation of specific substrate classes that are then differentially stabilized.","evidence":"Click-chemistry N-myristoylation assay, iTRAQ proteomics, liver conditional NMT1 knockout mouse, and PRM","pmids":["34136404"],"confidence":"Medium","gaps":["Biochemical role of POTEE in the NMT1 reaction not defined","Whether POTEE is itself myristoylated unaddressed"]},{"year":2023,"claim":"Resolved the molecular mechanism of POTEE-driven invasion, identifying it as a Rac1-SUMO1 effector that recruits TRIO-GEF to locally activate Rac1 at invadopodia.","evidence":"Co-localization, invadopodium formation and TRIO-GEF recruitment assays, with in vitro and in vivo proliferation/metastasis assays in breast cancer","pmids":["38098337"],"confidence":"Medium","gaps":["Binding interface between POTEE and Rac1-SUMO1 not mapped","Mechanism of TRIO-GEF recruitment unknown"]},{"year":2024,"claim":"Identified an upstream negative regulator, MARK1, that binds POTEE and suppresses its expression to limit proliferation in drug-resistant cells.","evidence":"Luciferase reporter binding assay, qRT-PCR, and overexpression/rescue in sorafenib-resistant hepatocellular carcinoma cells","pmids":["39534429"],"confidence":"Low","gaps":["Direct physical interaction not confirmed by biochemical methods","Mechanism of MARK1-mediated POTEE suppression undefined","Single-lab, single model system"]},{"year":2025,"claim":"Defined how POTEE protein abundance is post-translationally controlled and linked it to a metabolic output, showing LINC00667 directs TRIM33-mediated degradation of POTEE that regulates mitochondrial OXPHOS.","evidence":"CHIRP and RIP assays, cycloheximide chase, MG132 proteasome rescue, and OXPHOS complex western blotting with knockdown/overexpression","pmids":["40834976"],"confidence":"Medium","gaps":["Site of TRIM33-mediated ubiquitination on POTEE unmapped","Mechanism linking POTEE to OXPHOS complex expression unknown"]},{"year":null,"claim":"How POTEE physically links to its many effectors (Rac1, mTORC2, SPHK1, NMT1) through defined structural domains, and whether these activities reflect one unified scaffold function or distinct context-specific roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model or domain mapping for any interaction","No reconstitution of POTEE-dependent Rac1 or NMT1 activation in vitro","Unclear whether cancer-testis antigen expression confers a normal physiological function"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6]},{"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":[0,2,4,6]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8]}],"complexes":["mTORC2"],"partners":["MTOR","RICTOR","RAC1","TRIO","NMT1","TRIM33","MARK1","NEF"],"other_free_text":[]}},"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":30,"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":"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":12,"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":12,"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":10,"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":2,"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":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8515,"output_tokens":2426,"usd":0.030967,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9548,"output_tokens":2934,"usd":0.060545,"stage2_stop_reason":"end_turn"},"total_usd":0.091512,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"POTEE interacts with mTOR, RICTOR, and Rad51 in tumor-associated macrophages (TAMs), and this protein-protein interaction activates mTORC2, promoting cell invasion. siRNA-mediated knockdown of POTEE impairs cell survival in macrophages and TAMs.\",\n      \"method\": \"Co-immunoprecipitation (protein-protein interaction), siRNA knockdown with cell survival and invasion assays\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional siRNA knockdown in a single lab with multiple orthogonal assays\",\n      \"pmids\": [\"30420269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HIV-1 Nef directly interacts with POTEE (identified by pull-down and MALDI-TOF, validated by mammalian two-hybrid assay). The Nef-POTEE interaction recruits mTOR and RICTOR to form/activate mTORC2, which in turn activates AKT and PKC-α, leading to increased macrophage invasion and migration.\",\n      \"method\": \"Pull-down assay, MALDI-TOF mass spectrometry, mammalian two-hybrid assay, immunoprecipitation, cell invasion/migration assays\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pull-down, two-hybrid, Co-IP, functional assays) in a single lab\",\n      \"pmids\": [\"30391463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"POTEE promotes colorectal cancer cell growth, cell-cycle progression, and inhibits apoptosis via a POTEE/SPHK1/p65 signaling axis: overexpression of POTEE increases SPHK1 protein levels, which promotes phosphorylation and activation of p65.\",\n      \"method\": \"Microarray analysis, western blotting, overexpression and knockdown experiments, xenograft tumor model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional loss/gain-of-function with pathway validation by western blot, single lab\",\n      \"pmids\": [\"31723122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"POTEE is localized in the cytoplasm of colorectal carcinoma cells and promotes CRC migration, invasion, and epithelial-mesenchymal transition (EMT) by increasing activation of the small GTPases Rac1 and Cdc42.\",\n      \"method\": \"Immunohistochemistry (subcellular localization), qRT-PCR, western blotting, knockdown/overexpression, in vitro migration/invasion assays, in vivo xenograft, Rac1/Cdc42 activity assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional KD/OE with defined pathway readout (Rac1/Cdc42 activation), single lab, multiple orthogonal methods\",\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, and POTEE activates the PI3K/Akt/GSK-3β/β-catenin signaling pathway, as shown by downregulation of key pathway proteins upon POTEE knockdown.\",\n      \"method\": \"siRNA knockdown, GSK-3β inhibitor (Tideglusib), proliferation assays, western blotting of PI3K/Akt/GSK-3β/β-catenin pathway components\",\n      \"journal\": \"BioFactors (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional KD with pharmacological inhibitor rescue and pathway protein quantification, single lab\",\n      \"pmids\": [\"32589786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"N-myristoylation of POTEE targets (NDP and NUP protein categories) by NMT1 is dependent on POTEE; POTEE is required for NMT1-mediated N-myristoylation of these substrates. N-myristoylation differentially regulates protein stability: NDP proteins are destabilized (via RPL7A/HIST1H4H ubiquitin E3 ligase-mediated ubiquitination), while NUP proteins are stabilized (protected from ubiquitination by HBB).\",\n      \"method\": \"Click chemistry N-myristoylation assay, proteomics (iTRAQ), liver conditional NMT1 knockout mouse model, parallel reaction monitoring (PRM)\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — click chemistry enzymatic assay and in vivo KO model, single lab, multiple methods\",\n      \"pmids\": [\"34136404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"POTEE is a Rac1-SUMO1 effector: it co-localizes with SUMOylated Rac1 (Rac1-SUMO1) at invadopodia, recruits TRIO-GEF to activate Rac1 in the invadopodium, and thereby drives invadopodium formation, tumor cell proliferation, and metastasis in breast cancer cells in vitro and in vivo.\",\n      \"method\": \"Co-localization studies, invadopodium formation assays, TRIO-GEF recruitment assays, in vitro and in vivo tumor proliferation and metastasis assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assays with defined molecular interaction (Rac1-SUMO1/TRIO-GEF recruitment) in vitro and in vivo, single lab\",\n      \"pmids\": [\"38098337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MARK1 directly binds to POTEE (confirmed by luciferase reporter assay) and negatively regulates POTEE expression. Overexpression of MARK1 reduces POTEE levels and suppresses cell proliferation in sorafenib-resistant hepatocellular carcinoma cells; co-overexpression of POTEE reverses the MARK1-mediated suppression.\",\n      \"method\": \"Luciferase reporter assay, qRT-PCR, overexpression/rescue experiments in sorafenib-resistant cell models\",\n      \"journal\": \"Open medicine (Warsaw, Poland)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, luciferase binding assay and overexpression rescue without biochemical interaction confirmation\",\n      \"pmids\": [\"39534429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LINC00667 lncRNA directly binds POTEE protein (confirmed by CHIRP and RIP assays) and promotes TRIM33-mediated ubiquitination and proteasomal degradation of POTEE (demonstrated by cycloheximide chase and MG132 rescue). Degradation of POTEE decreases mitochondrial oxidative phosphorylation (OXPHOS) complex expression, while POTEE overexpression enhances OXPHOS activity.\",\n      \"method\": \"CHIRP assay, RIP assay, cycloheximide chase experiment, MG132 proteasome inhibitor treatment, OXPHOS complex expression by western blotting, knockdown/overexpression\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (CHIRP, RIP, CHX chase, proteasome inhibitor rescue) in a single lab identifying TRIM33 as E3 ligase and mitochondrial OXPHOS as functional output\",\n      \"pmids\": [\"40834976\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"POTEE is a cytoplasmic cancer-testis antigen that functions as an oncogenic scaffold/effector protein: it activates Rac1 (via recruitment of TRIO-GEF and interaction with SUMOylated Rac1) to drive invadopodium formation and metastasis; it signals through SPHK1/p65 and PI3K/Akt/GSK-3β/β-catenin axes to promote proliferation; it interacts with mTOR/RICTOR complexes (mTORC2) to activate AKT and PKC-α, enhancing macrophage and tumor cell invasion; it is required for NMT1-mediated N-myristoylation of specific substrates; it promotes mitochondrial OXPHOS and is subject to TRIM33-mediated ubiquitination/degradation regulated by LINC00667; and its expression is negatively regulated by MARK1 kinase through direct binding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"POTEE is a cytoplasmic oncogenic effector protein that promotes tumor cell proliferation, invasion, and metastasis across multiple cancer types by coupling to small-GTPase and growth-signaling pathways [#3, #6]. A central activity is activation of the Rho-family GTPases Rac1 and Cdc42: in colorectal carcinoma POTEE elevates Rac1/Cdc42 activity to drive migration, invasion, and epithelial-mesenchymal transition [#3], and in breast cancer it co-localizes with SUMOylated Rac1 (Rac1-SUMO1) at invadopodia where it recruits the TRIO guanine-nucleotide exchange factor to locally activate Rac1, driving invadopodium formation and metastasis [#6]. POTEE additionally engages mitogenic signaling through the SPHK1/p65 axis to promote cell-cycle progression and suppress apoptosis [#2] and through the PI3K/Akt/GSK-3\\u03b2/\\u03b2-catenin pathway to support proliferation [#4]. In macrophages it scaffolds mTOR and RICTOR to assemble and activate mTORC2, which activates AKT and PKC-\\u03b1 to enhance invasion, an interaction co-opted by HIV-1 Nef [#0, #1]. POTEE is also required for NMT1-mediated N-myristoylation of a defined set of substrate proteins [#5] and sustains mitochondrial oxidative phosphorylation, with its protein levels controlled by TRIM33-mediated ubiquitin-proteasomal degradation directed by the lncRNA LINC00667 [#8] and negatively regulated by MARK1 [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Established that POTEE acts as a scaffold linking to growth/survival signaling by physically assembling the mTORC2 machinery, answering how POTEE could influence invasion at the molecular level.\",\n      \"evidence\": \"Co-immunoprecipitation of POTEE with mTOR/RICTOR/Rad51 plus siRNA knockdown with survival and invasion assays in macrophages and TAMs; independently, pull-down/MALDI-TOF/two-hybrid showing HIV-1 Nef bridges POTEE to mTOR/RICTOR to activate AKT and PKC-\\u03b1\",\n      \"pmids\": [\"30420269\", \"30391463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect nature of the POTEE\\u2013mTOR/RICTOR contact not resolved\", \"Mapping of interaction domains absent\", \"Findings confined to macrophage/TAM context\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked POTEE to a defined proliferative signaling axis, showing it drives growth and blocks apoptosis through SPHK1-dependent p65 activation.\",\n      \"evidence\": \"Microarray, gain/loss-of-function, western blotting, and xenograft tumor model in colorectal cancer\",\n      \"pmids\": [\"31723122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which POTEE raises SPHK1 protein levels unknown\", \"No direct biochemical interaction demonstrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined POTEE's core motility function as activation of Rho-family GTPases and engagement of PI3K/Akt signaling, connecting it to invasion, EMT, and proliferation.\",\n      \"evidence\": \"Cytoplasmic localization by IHC, knockdown/overexpression with Rac1/Cdc42 activity assays and migration/invasion in CRC; siRNA knockdown plus GSK-3\\u03b2 inhibitor rescue and pathway western blotting in pancreatic cancer\",\n      \"pmids\": [\"32142855\", \"32589786\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How POTEE activates Rac1/Cdc42 not yet mechanistically defined\", \"Direct vs. indirect engagement of PI3K/Akt pathway unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a role for POTEE in protein lipidation, showing it is required for NMT1-mediated N-myristoylation of specific substrate classes that are then differentially stabilized.\",\n      \"evidence\": \"Click-chemistry N-myristoylation assay, iTRAQ proteomics, liver conditional NMT1 knockout mouse, and PRM\",\n      \"pmids\": [\"34136404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical role of POTEE in the NMT1 reaction not defined\", \"Whether POTEE is itself myristoylated unaddressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved the molecular mechanism of POTEE-driven invasion, identifying it as a Rac1-SUMO1 effector that recruits TRIO-GEF to locally activate Rac1 at invadopodia.\",\n      \"evidence\": \"Co-localization, invadopodium formation and TRIO-GEF recruitment assays, with in vitro and in vivo proliferation/metastasis assays in breast cancer\",\n      \"pmids\": [\"38098337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding interface between POTEE and Rac1-SUMO1 not mapped\", \"Mechanism of TRIO-GEF recruitment unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified an upstream negative regulator, MARK1, that binds POTEE and suppresses its expression to limit proliferation in drug-resistant cells.\",\n      \"evidence\": \"Luciferase reporter binding assay, qRT-PCR, and overexpression/rescue in sorafenib-resistant hepatocellular carcinoma cells\",\n      \"pmids\": [\"39534429\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Direct physical interaction not confirmed by biochemical methods\", \"Mechanism of MARK1-mediated POTEE suppression undefined\", \"Single-lab, single model system\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined how POTEE protein abundance is post-translationally controlled and linked it to a metabolic output, showing LINC00667 directs TRIM33-mediated degradation of POTEE that regulates mitochondrial OXPHOS.\",\n      \"evidence\": \"CHIRP and RIP assays, cycloheximide chase, MG132 proteasome rescue, and OXPHOS complex western blotting with knockdown/overexpression\",\n      \"pmids\": [\"40834976\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Site of TRIM33-mediated ubiquitination on POTEE unmapped\", \"Mechanism linking POTEE to OXPHOS complex expression unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How POTEE physically links to its many effectors (Rac1, mTORC2, SPHK1, NMT1) through defined structural domains, and whether these activities reflect one unified scaffold function or distinct context-specific roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model or domain mapping for any interaction\", \"No reconstitution of POTEE-dependent Rac1 or NMT1 activation in vitro\", \"Unclear whether cancer-testis antigen expression confers a normal physiological function\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 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, 2, 4, 6]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"mTORC2\"],\n    \"partners\": [\"MTOR\", \"RICTOR\", \"RAC1\", \"TRIO\", \"NMT1\", \"TRIM33\", \"MARK1\", \"NEF\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}