{"gene":"TSPAN8","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":1998,"finding":"TSPAN8 (D6.1A, rat homologue of CO-029) associates with α6β1 integrin at the cell membrane, demonstrated by Western blotting of D6.1A-positive tumor lines, and transfection of D6.1A cDNA into a low-metastasizing tumor line increased metastatic potential accompanied by consumption coagulopathy.","method":"cDNA transfection, Western blotting, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP/Western blot with functional gain-of-function transfection, single lab","pmids":["9531564"],"is_preprint":false},{"year":2005,"finding":"CO-029 (TSPAN8) co-localizes and co-immunoprecipitates with α6β4 integrin in human pancreatic adenocarcinoma cells; protein kinase C activation strengthens this association, leading to internalization of the integrin-tetraspanin complex, decreased laminin-5 adhesion, and increased cell migration.","method":"Co-immunoprecipitation, immunofluorescence colocalization, PKC activation assay, migration assay","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal Co-IP with functional consequence (migration), single lab","pmids":["15837731"],"is_preprint":false},{"year":2006,"finding":"CO-029/D6.1A (TSPAN8) expressed on tumor cells and their exosomes induces angiogenesis by stimulating MMP and uPA secretion, VEGF expression in fibroblasts, and VEGF receptor upregulation in sprouting endothelium; anti-D6.1A antibody completely blocked angiogenesis.","method":"In vivo angiogenesis assay, in vitro endothelial cell branching assay, antibody blocking, gene expression analysis","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional readouts in vivo and in vitro, single lab","pmids":["16849554"],"is_preprint":false},{"year":2010,"finding":"Tspan8 selectively recruits proteins (including CD106 and CD49d) and mRNA into exosomes; exosomes bearing a Tspan8-CD49d complex preferentially bind and are internalized by endothelial cells, inducing VEGF-independent upregulation of angiogenesis-related genes and enhanced EC proliferation, migration, and sprouting.","method":"Exosome isolation and characterization, co-immunoprecipitation, gene expression profiling, EC functional assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, functional assays, gene expression) with specific mechanistic insight, replicated across studies","pmids":["20124479"],"is_preprint":false},{"year":2010,"finding":"Activation-induced Tspan8 internalization is faster than CD9 internalization and relies on association of the Tspan8 N-terminal region with intersectin-2 (a clathrin-coated pit component); PMA activation promotes recruitment of CD49d into a Tspan8-intersectin-2-CD49d-clathrin complex in cholesterol-depletion-resistant microdomains, promoting cell migration while reducing matrix and cell adhesion.","method":"Tspan8-chimera internalization assays, co-immunoprecipitation, cholesterol depletion, confocal microscopy, migration assays","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic dissection with chimera mutants and Co-IP, single lab","pmids":["20937409"],"is_preprint":false},{"year":2010,"finding":"CO-029/Tspan8 directly interacts with E-cadherin (shown by chemical cross-linking and co-immunoprecipitation) and cooperates with p120-catenin to regulate colon carcinoma cell motility via a switch in signaling between collagen-binding integrins α1β1 and α2β1.","method":"Chemical cross-linking, co-immunoprecipitation, siRNA knockdown, migration assay, antibody blocking","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 — direct interaction by chemical cross-linking plus Co-IP, functional rescue/knockdown with specific integrin-switch mechanism","pmids":["20858717"],"is_preprint":false},{"year":2012,"finding":"CO-029 (TSPAN8) knockdown in HT29 colon cancer cells reduces migration and increases integrin-dependent cell-matrix adhesion on laminin, upregulates cell surface levels of laminin-binding integrin α3β1 and α5β1, decreases CD44, and increases cadherin-mediated cell-cell adhesion while reducing MelCAM levels.","method":"siRNA knockdown, migration assay, flow cytometry for integrin surface levels, adhesion assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — KD with multiple specific molecular readouts, single lab","pmids":["22679508"],"is_preprint":false},{"year":2013,"finding":"Tspan8 knockdown in rat pancreatic adenocarcinoma ASML cells increases adhesion due to CD151 associating with α3 integrin; Tspan8 recruits β4 integrin into Tspan8 complexes with accompanying β4 phosphorylation, Src recruitment, FAK and Ras activation, driving motility over adhesion; CD151 associates more readily with MMP13 and MMP9 than Tspan8.","method":"Stable knockdown, co-immunoprecipitation, phosphorylation assays, signaling pathway analysis, invasion/migration assays","journal":"European journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — KD with defined signaling mechanism, multiple readouts, single lab","pmids":["23683890"],"is_preprint":false},{"year":2015,"finding":"Exosomal Tspan8 and CD151 support matrix degradation through tetraspanin-integrin and tetraspanin-protease associations; exosomes deficient in CD151/Tspan8 have reduced binding/uptake and fail to reprogram stroma, hematopoietic cells, or drive EMT gene expression in poorly metastatic cells.","method":"Stable knockdown, exosome isolation, functional reconstitution, co-immunoprecipitation","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — systematic knockdown with mechanistic exosome rescue, single lab","pmids":["25544774"],"is_preprint":false},{"year":2015,"finding":"TM4SF3/TSPAN8 is localized not only to the plasma membrane but also to the nucleus of prostate cancer cells; this nuclear localization depends on androgen receptor (AR) nuclear localization. TM4SF3 directly interacts with AR, and this interaction stabilizes both proteins by reducing ubiquitin-proteasomal degradation; TM4SF3 is required for androgen-dependent gene expression and cancer cell proliferation.","method":"Co-immunoprecipitation, nuclear fractionation, immunofluorescence, siRNA knockdown, in vitro interaction assay, gene expression analysis","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 1-2 — direct interaction confirmed in vitro and by Co-IP, nuclear localization by fractionation and IF, functional knockdown with multiple readouts","pmids":["26649804"],"is_preprint":false},{"year":2015,"finding":"LSD1 (histone demethylase) epigenetically upregulates TSPAN8 expression by reducing H3K9me2 occupancy on the TSPAN8 promoter in colorectal cancer cells; TSPAN8 promotes EMT in an LSD1-dependent manner.","method":"ChIP, siRNA knockdown, LSD1 inhibitor treatment, RT-PCR, Western blot, migration assay","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP identifies epigenetic writer mechanism with functional downstream EMT readout, single lab","pmids":["31790687"],"is_preprint":false},{"year":2018,"finding":"In Tspan8/CD151 knockout mice, impaired tumor dissemination is associated with distorted tetraspanin associations with integrins and CAM, and missing Tspan8/CD151-promoted recruitment of proteases; host Tspan8 in exosomes contributes to angiogenesis through association with GPCR and RTK in endothelial cells.","method":"Tspan8 knockout mice, tumor implantation, exosome coculture, co-immunoprecipitation, in vitro rescue experiments","journal":"Journal of experimental & clinical cancer research","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout model with mechanistic rescue experiments, multiple cell types tested","pmids":["30541597"],"is_preprint":false},{"year":2019,"finding":"TSPAN8 interacts with PTCH1 and inhibits degradation of the SHH/PTCH1 complex by recruiting deubiquitinating enzyme ATXN3, thereby stabilizing the complex and enabling SMO translocation to cilia, activating downstream Hedgehog signaling and promoting cancer stem cell properties including NANOG/OCT4 expression and chemoresistance.","method":"Co-immunoprecipitation, ubiquitination assay, confocal microscopy (cilia localization), siRNA knockdown, in vivo tumor formation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic Co-IP identifying deubiquitinase recruitment, functional epistasis confirmed by multiple assays in vitro and in vivo","pmids":["31253779"],"is_preprint":false},{"year":2019,"finding":"Tspan8 promotes breast cancer cell E-cadherin upregulation and Twist/p120-catenin/β-catenin target gene downregulation (resembling MET), and co-immunoprecipitation showed Tspan8 and p120-catenin interact; Tspan8 mediates a several-fold increase in extracellular vesicle numbers in cell culture and in vivo.","method":"Co-immunoprecipitation, EV quantification, Western blot, cell behavior assays","journal":"The Journal of pathology","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP with partial mechanistic follow-up, single lab","pmids":["30982971"],"is_preprint":false},{"year":2019,"finding":"β-catenin stabilization occurs downstream of Tspan8 expression in melanoma; β-catenin in turn directly transcriptionally activates Tspan8, forming a positive feedback loop that sustains Tspan8-driven melanoma invasion.","method":"Tspan8 overexpression/knockdown, β-catenin reporter assay, ChIP, invasion assay, in vivo transgenic mouse model","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP confirms direct transcriptional activation, feedback loop validated in vivo and in vitro, single lab","pmids":["30679790"],"is_preprint":false},{"year":2020,"finding":"Tspan8+ melanoma cells cooperate with surrounding keratinocytes to activate keratinocyte-derived proMMP-9, degrade collagen IV, and achieve dermal colonization; this involves elevated MMP-3 and low TIMP-1 levels; anti-Tspan8 antibody reduces proMMP-9 activation and dermal invasion.","method":"3D skin reconstruct model, MMP activity assay, antibody blocking, siRNA knockdown","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — functional reconstitution in validated 3D model, multiple mechanistic readouts, antibody validation, single lab","pmids":["32455575"],"is_preprint":false},{"year":2022,"finding":"EGFR signaling induces AKT-mediated phosphorylation of TSPAN8 at Ser129, enabling TSPAN8 binding to 14-3-3θ and importin-β1, which drives TSPAN8 nuclear translocation; in the nucleus, phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and transcription of MYC, BCL2, MMP9 and other oncogenes.","method":"Co-immunoprecipitation, phospho-site mutagenesis, nuclear fractionation, ChIP, reporter assays, in vitro kinase assay, antibody blocking in vivo","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis, ChIP, Co-IP, and in vivo validation with anti-TSPAN8 antibody, multiple orthogonal methods","pmids":["35197608"],"is_preprint":false},{"year":2023,"finding":"TM4SF3/TSPAN8 physically interacts with AR-V7 (in addition to AR); interaction of TM4SF3 with AR or AR-V7 results in mutual deubiquitination and stabilization of both proteins; nuclear TM4SF3 is co-recruited to promoters of AR/AR-V7-regulated genes and is required for their transcriptional activation.","method":"Co-immunoprecipitation, ubiquitination assay, ChIP, nuclear fractionation, siRNA knockdown, domain mapping","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 1-2 — Co-IP with ubiquitination assay, ChIP showing promoter co-recruitment, functional knockdown, multiple readouts","pmids":["36951301"],"is_preprint":false},{"year":2023,"finding":"Tspan8 coalesces with lipid rafts and facilitates IFN-γR1 localization at or near lipid rafts; Tspan8 silencing impairs lipid raft-mediated but promotes clathrin-mediated endocytosis of IFN-γR1, leading to increased STAT1 signaling and intestinal epithelial permeability.","method":"siRNA knockdown, lipid raft fractionation, endocytosis assay (clathrin vs lipid raft), STAT1 signaling readout, barrier permeability assay","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic dissection of two endocytosis routes with functional consequence on signaling, single lab","pmids":["37204469"],"is_preprint":false},{"year":2023,"finding":"Tspan8 associates with endothelin-converting enzyme ECE1 and amplifies its enzymatic activity in converting big-ET1 to endothelin-1, demonstrated by transduction of Tspan8 into Isreco1 cells and by comparing ileum tissue of tspan8 knockout vs. wild-type mice.","method":"Mass spectrometry, Western blot co-immunoprecipitation, ECE1 activity assay, tspan8 knockout mouse tissue comparison","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — MS-identified partner with enzymatic activity assay confirmed in KO mouse tissue, single lab","pmids":["37835445"],"is_preprint":false},{"year":2024,"finding":"TSPAN8 promotes phosphorylation of E3 ligase RBBP6 at Ser772 by recruiting MAPK11, inducing SIRT6 protein degradation; SIRT6 downregulation subsequently upregulates GLS1 and PYCR1, causing TSPAN8+ myofibroblastic CAFs to secrete aspartate and proline that support breast cancer cell outgrowth.","method":"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, SIRT6 activity assay, metabolite secretion measurement, in vivo xenograft","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1-2 — mechanistic Co-IP identifying kinase recruitment and substrate phosphorylation, downstream metabolic consequence validated in vivo","pmids":["38569015"],"is_preprint":false},{"year":2024,"finding":"MDM2 acts as the common E3 ubiquitin ligase for TM4SF3/TSPAN8, AR, and AR-V7; MDM2 inhibition elevated all three proteins by reducing their ubiquitination; TM4SF3 interaction with AR or AR-V7 results in mutual deubiquitination and prevents proteasomal degradation.","method":"siRNA depletion of E3 ligases, MDM2 pharmacological inhibitor, ubiquitination assay, protein stability assay","journal":"Endocrine oncology","confidence":"Medium","confidence_rationale":"Tier 2 — identification of specific E3 ligase by systematic siRNA screen and pharmacological inhibition with ubiquitination readout, single lab","pmids":["38410785"],"is_preprint":false},{"year":2009,"finding":"In Xenopus, tm4sf3 (TSPAN8 ortholog) is specifically expressed in the ventral pancreas at the fusion junction; morpholino-mediated knockdown inhibits dorsal-ventral pancreatic bud fusion and acinar cell differentiation, while overexpression promotes annular pancreas development.","method":"Morpholino knockdown, mRNA overexpression, Xenopus embryo model, in situ hybridization","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 — loss- and gain-of-function in a vertebrate model with defined developmental phenotype, single lab","pmids":["19403659"],"is_preprint":false},{"year":2021,"finding":"TSPAN8-high spermatogonia in mouse testis are enriched for spermatogonial stem cells (SSCs), as demonstrated by transplantation studies showing that TSPAN8-high subpopulations have greater stem cell activity than TSPAN8-low progenitor spermatogonia.","method":"Flow cytometry cell sorting, spermatogonial transplantation assay, RNA-seq, ChIP-seq, Methyl-seq","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — transplantation (gold-standard SSC assay) directly links TSPAN8 expression to SSC identity, single lab","pmids":["27733379"],"is_preprint":false},{"year":2021,"finding":"TSPAN8 promotes cancer cell stemness by activating Sonic Hedgehog signaling through exosomal transfer; CSC-derived TSPAN8-enriched exosomes activate Hh signaling in non-stem cancer cells, increasing their clonogenic ability, invasiveness, and chemoresistance in PDAC.","method":"Single-cell RNA sequencing, exosomal profiling, functional assays (clonogenicity, invasion, chemoresistance), in vivo tumor growth","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — exosome transfer experiments with pathway-specific functional readouts, in vivo validation, single lab","pmids":["40251391"],"is_preprint":false},{"year":2019,"finding":"TSPAN8 directly interacts with β-catenin (by Co-IP), enhances β-catenin protein expression, and promotes colorectal cancer stemness; β-catenin in turn directly binds the TSPAN8 promoter (by ChIP) and enhances TSPAN8 transcription, forming a positive regulatory loop.","method":"Co-immunoprecipitation, ChIP, siRNA knockdown, sphere formation assay, Western blot","journal":"Medical science monitor","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus ChIP establish bidirectional regulation with functional stemness readout, single lab","pmids":["31838484"],"is_preprint":false},{"year":2022,"finding":"TSPAN8 forms a complex with Rictor (a component of mTORC2) and overexpression of TSPAN8 suppresses high-glucose-induced autophagy and apoptosis in kidney cells in an mTOR-dependent manner.","method":"Co-immunoprecipitation, TSPAN8 overexpression, mTOR inhibitor treatment, apoptosis/autophagy assays","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP identifying mTORC2 complex, functional assay without detailed mechanistic follow-up","pmids":["35904232"],"is_preprint":false}],"current_model":"TSPAN8 is a tetraspanin that functions as a membrane organizer forming complexes with integrins (α6β4, α6β1, α3β1), E-cadherin, EGFR, PTCH1, ECE1, AR, and mTORC2; it controls cell adhesion, migration, and invasion by regulating integrin-adhesion signaling and MMP activation, drives angiogenesis via selective loading of protein/mRNA cargo into exosomes bearing Tspan8-CD49d complexes, promotes cancer stemness by recruiting deubiquitinase ATXN3 to stabilize SHH/PTCH1 and activate Hedgehog signaling, undergoes EGFR-AKT-dependent phosphorylation at Ser129 enabling 14-3-3θ/importin-β1-mediated nuclear translocation where it co-activates STAT3 transcription, stabilizes AR/AR-V7 through mutual deubiquitination in prostate cancer, and regulates IFN-γR1 endocytosis route (lipid raft vs. clathrin) to control intestinal epithelial barrier integrity."},"narrative":{"teleology":[{"year":1998,"claim":"Establishing TSPAN8 as a metastasis-associated tetraspanin that partners with integrins: transfection of D6.1A into a low-metastasizing line proved that TSPAN8 expression is sufficient to increase metastatic potential and demonstrated the first integrin partner (α6β1).","evidence":"cDNA transfection and Co-IP in rat tumor lines","pmids":["9531564"],"confidence":"Medium","gaps":["No reciprocal validation of integrin interaction with purified proteins","Mechanism linking integrin association to metastasis not defined"]},{"year":2005,"claim":"Defining how TSPAN8–integrin complexes regulate cell behavior: PKC-induced strengthening of the TSPAN8–α6β4 complex caused integrin internalization, decreased laminin adhesion, and increased migration, linking tetraspanin–integrin dynamics to a motility switch.","evidence":"Co-IP, PKC activation, migration assay in human pancreatic carcinoma cells","pmids":["15837731"],"confidence":"Medium","gaps":["PKC substrate specificity on TSPAN8 or integrin not identified","In vivo relevance not tested"]},{"year":2006,"claim":"Revealing TSPAN8 as a driver of angiogenesis: tumor-cell and exosome-expressed TSPAN8 induced MMP/uPA secretion and VEGF/VEGFR upregulation, establishing a non-cell-autonomous pro-angiogenic role.","evidence":"In vivo angiogenesis assay with anti-D6.1A antibody blocking","pmids":["16849554"],"confidence":"Medium","gaps":["Direct molecular mechanism linking TSPAN8 to VEGF induction unclear","Exosome-specific versus cell-surface contribution not separated"]},{"year":2009,"claim":"Demonstrating a developmental role for TSPAN8: morpholino knockdown in Xenopus showed TSPAN8 is required for dorsal–ventral pancreatic bud fusion and acinar differentiation, broadening its biology beyond cancer.","evidence":"Morpholino knockdown and mRNA overexpression in Xenopus embryos","pmids":["19403659"],"confidence":"Medium","gaps":["Mammalian pancreatic development role not confirmed","Downstream signaling pathway mediating bud fusion unknown"]},{"year":2010,"claim":"Elucidating TSPAN8's role in exosome cargo selection and VEGF-independent angiogenesis: Tspan8–CD49d complexes selectively recruited proteins and mRNA into exosomes that were preferentially taken up by endothelial cells, activating angiogenic gene programs independently of VEGF.","evidence":"Exosome isolation, Co-IP, gene expression profiling, EC functional assays","pmids":["20124479"],"confidence":"High","gaps":["Sorting signal within TSPAN8 for exosome loading not mapped","Whether TSPAN8 functions as cargo receptor or scaffold not resolved"]},{"year":2010,"claim":"Dissecting TSPAN8 internalization and adhesion-complex remodeling: TSPAN8's N-terminal region recruits intersectin-2 into clathrin-dependent complexes with CD49d, explaining how activation-induced TSPAN8 endocytosis couples to reduced adhesion and enhanced migration, and how TSPAN8 interacts with E-cadherin and p120-catenin to switch between collagen-binding integrins α1β1 and α2β1.","evidence":"Chimera internalization assays, chemical cross-linking, Co-IP, siRNA knockdown, migration assays","pmids":["20937409","20858717"],"confidence":"High","gaps":["Structural basis of TSPAN8–E-cadherin interaction not resolved","Whether intersectin-2 route and E-cadherin complex operate in the same cell context unclear"]},{"year":2013,"claim":"Mapping TSPAN8 signaling downstream of integrin β4: TSPAN8 recruits β4 integrin, promotes its phosphorylation and Src/FAK/Ras activation, shifting cells from adhesion to motility, while CD151 preferentially associates with MMPs—establishing non-redundant tetraspanin functions.","evidence":"Stable knockdown, Co-IP, phosphorylation and signaling assays in rat pancreatic adenocarcinoma","pmids":["23683890"],"confidence":"Medium","gaps":["Kinase responsible for β4 phosphorylation not identified","Whether TSPAN8 and CD151 compete for the same integrin pools unclear"]},{"year":2015,"claim":"Discovering TSPAN8 nuclear localization and AR stabilization: TSPAN8 was found in the nucleus of prostate cancer cells in an AR-dependent manner, directly interacting with AR and reducing its ubiquitin-proteasomal degradation, establishing TSPAN8 as a transcriptional co-regulator.","evidence":"Co-IP, nuclear fractionation, in vitro interaction assay, siRNA knockdown","pmids":["26649804"],"confidence":"High","gaps":["Deubiquitinase mediating AR stabilization by TSPAN8 not identified at this point","Mechanism of TSPAN8 nuclear import not defined"]},{"year":2018,"claim":"In vivo genetic validation: Tspan8/CD151 knockout mice showed impaired tumor dissemination with distorted tetraspanin–integrin–protease networks, and host-derived Tspan8-containing exosomes promoted angiogenesis through GPCR/RTK associations in endothelial cells.","evidence":"Knockout mice, tumor implantation, exosome coculture, Co-IP, rescue experiments","pmids":["30541597"],"confidence":"High","gaps":["Single vs. double KO contributions not fully deconvoluted","Identity of specific GPCR/RTK partners on endothelial cells not determined"]},{"year":2019,"claim":"Linking TSPAN8 to Hedgehog pathway and cancer stemness: TSPAN8 recruited deubiquitinase ATXN3 to stabilize the SHH/PTCH1 complex, enabling SMO ciliary translocation and downstream Hh signaling that drove NANOG/OCT4 expression and chemoresistance; a β-catenin–TSPAN8 positive feedback loop was simultaneously identified in melanoma and colorectal cancer.","evidence":"Co-IP, ubiquitination assay, confocal cilia imaging, ChIP for β-catenin on TSPAN8 promoter, in vivo tumor formation","pmids":["31253779","30679790","31838484"],"confidence":"High","gaps":["Whether ATXN3 recruitment is direct or scaffold-mediated not resolved","Intersection between Hh and β-catenin feedback loops not explored"]},{"year":2022,"claim":"Defining the nuclear import mechanism: EGFR–AKT phosphorylates TSPAN8 at Ser129, creating a 14-3-3θ binding site that enables importin-β1-mediated nuclear translocation; nuclear phospho-TSPAN8 enhances STAT3 chromatin occupancy at MYC, BCL2, and MMP9 promoters.","evidence":"In vitro kinase assay, phospho-site mutagenesis, ChIP, nuclear fractionation, anti-TSPAN8 antibody in vivo","pmids":["35197608"],"confidence":"High","gaps":["Whether nuclear TSPAN8–STAT3 complex requires additional co-factors unknown","Relationship between AR-dependent and EGFR-dependent nuclear import not clarified"]},{"year":2023,"claim":"Extending the AR co-regulator role: TSPAN8 was shown to interact with AR-V7 in addition to full-length AR, with mutual deubiquitination enabling both proteins' stabilization and co-recruitment to AR/AR-V7-regulated gene promoters; separately, TSPAN8 was found to direct IFN-γR1 into lipid-raft endocytosis to restrain STAT1 signaling and maintain intestinal barrier integrity, and to complex with ECE1 to amplify endothelin-1 production.","evidence":"Co-IP, ubiquitination assay, ChIP, lipid raft fractionation, ECE1 activity assay in Tspan8 KO mouse tissue","pmids":["36951301","37204469","37835445"],"confidence":"High","gaps":["Deubiquitinase responsible for AR/TSPAN8 mutual stabilization still unidentified","Whether ECE1 activation is direct or via membrane microdomain organization not established"]},{"year":2024,"claim":"Revealing a stromal metabolic reprogramming function: in myofibroblastic cancer-associated fibroblasts, TSPAN8 recruits MAPK11 to phosphorylate E3 ligase RBBP6 at Ser772, triggering SIRT6 degradation and upregulating GLS1/PYCR1-dependent aspartate and proline secretion that feeds tumor cell outgrowth; MDM2 was identified as the E3 ligase mediating ubiquitination of TSPAN8, AR, and AR-V7.","evidence":"Co-IP, phosphorylation assay, metabolite measurement, in vivo xenograft; systematic E3 ligase siRNA screen with MDM2 inhibitor","pmids":["38569015","38410785"],"confidence":"High","gaps":["Whether MAPK11 recruitment is a general TSPAN8 function or CAF-specific unknown","Structural basis of TSPAN8–MDM2 interaction not resolved"]},{"year":null,"claim":"Key unresolved questions include: the structural basis of TSPAN8's multivalent scaffolding across integrins, receptors, and nuclear partners; whether the EGFR–AKT and AR-dependent nuclear import pathways converge or operate independently; the identity of the deubiquitinase(s) mediating mutual AR/TSPAN8 stabilization; and how TSPAN8 cargo-sorting specificity in exosomes is encoded.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of TSPAN8 in complex with any partner","No reconstitution of TSPAN8-dependent exosome sorting with purified components","Nuclear versus membrane pool regulation not mechanistically separated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4,5,12,16,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12,18,19]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[9,16,17]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,5,18]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,16,17]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,8,24]}],"pathway":[{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[5,6,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,16,18,25]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,3,7,8,11,15,20]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,8,24]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[22]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[7,15]}],"complexes":["TSPAN8–integrin α6β4 complex","TSPAN8–E-cadherin–p120-catenin complex","TSPAN8–AR/AR-V7 complex","TSPAN8–PTCH1–ATXN3 complex"],"partners":["ITGB4","ITGA6","CDH1","CTNND1","AR","PTCH1","ATXN3","STAT3"],"other_free_text":[]},"mechanistic_narrative":"TSPAN8 is a tetraspanin that organizes membrane microdomains to coordinate integrin-mediated adhesion, cell migration, extracellular vesicle biogenesis, and receptor signaling across epithelial, endothelial, and stromal compartments. At the plasma membrane, TSPAN8 forms complexes with integrins (α6β4, α6β1, α3β1), E-cadherin, and p120-catenin to regulate adhesion–motility switching through Src/FAK/Ras activation and MMP-dependent matrix remodeling, and it selectively loads cargo into exosomes that drive VEGF-independent angiogenesis and paracrine Hedgehog pathway activation in recipient cells [PMID:15837731, PMID:20858717, PMID:23683890, PMID:20124479, PMID:31253779]. TSPAN8 also undergoes EGFR–AKT-dependent phosphorylation at Ser129, enabling 14-3-3θ/importin-β1-mediated nuclear translocation where it co-activates STAT3 target genes, and in prostate cancer it stabilizes AR/AR-V7 through mutual protection from MDM2-mediated ubiquitination [PMID:35197608, PMID:26649804, PMID:36951301, PMID:38410785]. Beyond tumor biology, TSPAN8 directs IFN-γR1 into lipid-raft endocytic routes to restrain STAT1 signaling and maintain intestinal epithelial barrier integrity, interacts with ECE1 to amplify endothelin-1 production, and is required for ventral–dorsal pancreatic bud fusion during Xenopus development [PMID:37204469, PMID:37835445, PMID:19403659]."},"prefetch_data":{"uniprot":{"accession":"P19075","full_name":"Tetraspanin-8","aliases":["Transmembrane 4 superfamily member 3","Tumor-associated antigen CO-029"],"length_aa":237,"mass_kda":26.0,"function":"Structural component of specialized membrane microdomains known as tetraspanin-enriched microdomains (TERMs), which act as platforms for receptor clustering and signaling (PubMed:27180357, PubMed:36078095). Participates thereby in diverse biological functions such as cell signal transduction, migration and protein trafficking (PubMed:25761241). Promotes ADAM17-mediated TNF processing through recruitment of ADAM17 to tetraspanin-enriched micro-domains (TEMs) (PubMed:36078095). Forms a complex with RICTOR and integrin alpha3/ITGA3 to mediate mTORC2 activation and AKT1 phosphorylation leading to cell migration (PubMed:25761241). Reduces apoptosis and autophagy induced by high glucose levels through forming a complex with mTOR and RICTOR (PubMed:35904232). Contributes to the maintenance of intestinal epithelial barrier and plays a role in the regulation of intestine inflammation by switching interferon gamma receptor 1/IFNGR1 from clathrin-dependent to lipid raft-dependent endocytosis route to limit STAT1 activation magnitude and duration (PubMed:37204469). Acts as a modulator of the endothelin axis by associating with endothelin converting enzyme ECE1 and regulating its activity of conversion of the endothelin-1 precursor to endothelin (PubMed:37835445)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P19075/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSPAN8","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":[],"url":"https://opencell.sf.czbiohub.org/search/TSPAN8","total_profiled":1310},"omim":[{"mim_id":"608212","title":"IMMUNITY-RELATED GTPase M; IRGM","url":"https://www.omim.org/entry/608212"},{"mim_id":"600769","title":"TETRASPANIN 8; TSPAN8","url":"https://www.omim.org/entry/600769"},{"mim_id":"222100","title":"TYPE 1 DIABETES 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States","date":"2025-09-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.27.672753","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":35646,"output_tokens":6245,"usd":0.100307},"stage2":{"model":"claude-opus-4-6","input_tokens":9973,"output_tokens":3965,"usd":0.223485},"total_usd":0.323792,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"TSPAN8 (D6.1A, rat homologue of CO-029) associates with α6β1 integrin at the cell membrane, demonstrated by Western blotting of D6.1A-positive tumor lines, and transfection of D6.1A cDNA into a low-metastasizing tumor line increased metastatic potential accompanied by consumption coagulopathy.\",\n      \"method\": \"cDNA transfection, Western blotting, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP/Western blot with functional gain-of-function transfection, single lab\",\n      \"pmids\": [\"9531564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CO-029 (TSPAN8) co-localizes and co-immunoprecipitates with α6β4 integrin in human pancreatic adenocarcinoma cells; protein kinase C activation strengthens this association, leading to internalization of the integrin-tetraspanin complex, decreased laminin-5 adhesion, and increased cell migration.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, PKC activation assay, migration assay\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with functional consequence (migration), single lab\",\n      \"pmids\": [\"15837731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CO-029/D6.1A (TSPAN8) expressed on tumor cells and their exosomes induces angiogenesis by stimulating MMP and uPA secretion, VEGF expression in fibroblasts, and VEGF receptor upregulation in sprouting endothelium; anti-D6.1A antibody completely blocked angiogenesis.\",\n      \"method\": \"In vivo angiogenesis assay, in vitro endothelial cell branching assay, antibody blocking, gene expression analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional readouts in vivo and in vitro, single lab\",\n      \"pmids\": [\"16849554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Tspan8 selectively recruits proteins (including CD106 and CD49d) and mRNA into exosomes; exosomes bearing a Tspan8-CD49d complex preferentially bind and are internalized by endothelial cells, inducing VEGF-independent upregulation of angiogenesis-related genes and enhanced EC proliferation, migration, and sprouting.\",\n      \"method\": \"Exosome isolation and characterization, co-immunoprecipitation, gene expression profiling, EC functional assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, functional assays, gene expression) with specific mechanistic insight, replicated across studies\",\n      \"pmids\": [\"20124479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Activation-induced Tspan8 internalization is faster than CD9 internalization and relies on association of the Tspan8 N-terminal region with intersectin-2 (a clathrin-coated pit component); PMA activation promotes recruitment of CD49d into a Tspan8-intersectin-2-CD49d-clathrin complex in cholesterol-depletion-resistant microdomains, promoting cell migration while reducing matrix and cell adhesion.\",\n      \"method\": \"Tspan8-chimera internalization assays, co-immunoprecipitation, cholesterol depletion, confocal microscopy, migration assays\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection with chimera mutants and Co-IP, single lab\",\n      \"pmids\": [\"20937409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CO-029/Tspan8 directly interacts with E-cadherin (shown by chemical cross-linking and co-immunoprecipitation) and cooperates with p120-catenin to regulate colon carcinoma cell motility via a switch in signaling between collagen-binding integrins α1β1 and α2β1.\",\n      \"method\": \"Chemical cross-linking, co-immunoprecipitation, siRNA knockdown, migration assay, antibody blocking\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct interaction by chemical cross-linking plus Co-IP, functional rescue/knockdown with specific integrin-switch mechanism\",\n      \"pmids\": [\"20858717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CO-029 (TSPAN8) knockdown in HT29 colon cancer cells reduces migration and increases integrin-dependent cell-matrix adhesion on laminin, upregulates cell surface levels of laminin-binding integrin α3β1 and α5β1, decreases CD44, and increases cadherin-mediated cell-cell adhesion while reducing MelCAM levels.\",\n      \"method\": \"siRNA knockdown, migration assay, flow cytometry for integrin surface levels, adhesion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with multiple specific molecular readouts, single lab\",\n      \"pmids\": [\"22679508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tspan8 knockdown in rat pancreatic adenocarcinoma ASML cells increases adhesion due to CD151 associating with α3 integrin; Tspan8 recruits β4 integrin into Tspan8 complexes with accompanying β4 phosphorylation, Src recruitment, FAK and Ras activation, driving motility over adhesion; CD151 associates more readily with MMP13 and MMP9 than Tspan8.\",\n      \"method\": \"Stable knockdown, co-immunoprecipitation, phosphorylation assays, signaling pathway analysis, invasion/migration assays\",\n      \"journal\": \"European journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with defined signaling mechanism, multiple readouts, single lab\",\n      \"pmids\": [\"23683890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Exosomal Tspan8 and CD151 support matrix degradation through tetraspanin-integrin and tetraspanin-protease associations; exosomes deficient in CD151/Tspan8 have reduced binding/uptake and fail to reprogram stroma, hematopoietic cells, or drive EMT gene expression in poorly metastatic cells.\",\n      \"method\": \"Stable knockdown, exosome isolation, functional reconstitution, co-immunoprecipitation\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic knockdown with mechanistic exosome rescue, single lab\",\n      \"pmids\": [\"25544774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TM4SF3/TSPAN8 is localized not only to the plasma membrane but also to the nucleus of prostate cancer cells; this nuclear localization depends on androgen receptor (AR) nuclear localization. TM4SF3 directly interacts with AR, and this interaction stabilizes both proteins by reducing ubiquitin-proteasomal degradation; TM4SF3 is required for androgen-dependent gene expression and cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, nuclear fractionation, immunofluorescence, siRNA knockdown, in vitro interaction assay, gene expression analysis\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct interaction confirmed in vitro and by Co-IP, nuclear localization by fractionation and IF, functional knockdown with multiple readouts\",\n      \"pmids\": [\"26649804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"LSD1 (histone demethylase) epigenetically upregulates TSPAN8 expression by reducing H3K9me2 occupancy on the TSPAN8 promoter in colorectal cancer cells; TSPAN8 promotes EMT in an LSD1-dependent manner.\",\n      \"method\": \"ChIP, siRNA knockdown, LSD1 inhibitor treatment, RT-PCR, Western blot, migration assay\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP identifies epigenetic writer mechanism with functional downstream EMT readout, single lab\",\n      \"pmids\": [\"31790687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Tspan8/CD151 knockout mice, impaired tumor dissemination is associated with distorted tetraspanin associations with integrins and CAM, and missing Tspan8/CD151-promoted recruitment of proteases; host Tspan8 in exosomes contributes to angiogenesis through association with GPCR and RTK in endothelial cells.\",\n      \"method\": \"Tspan8 knockout mice, tumor implantation, exosome coculture, co-immunoprecipitation, in vitro rescue experiments\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout model with mechanistic rescue experiments, multiple cell types tested\",\n      \"pmids\": [\"30541597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TSPAN8 interacts with PTCH1 and inhibits degradation of the SHH/PTCH1 complex by recruiting deubiquitinating enzyme ATXN3, thereby stabilizing the complex and enabling SMO translocation to cilia, activating downstream Hedgehog signaling and promoting cancer stem cell properties including NANOG/OCT4 expression and chemoresistance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, confocal microscopy (cilia localization), siRNA knockdown, in vivo tumor formation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic Co-IP identifying deubiquitinase recruitment, functional epistasis confirmed by multiple assays in vitro and in vivo\",\n      \"pmids\": [\"31253779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Tspan8 promotes breast cancer cell E-cadherin upregulation and Twist/p120-catenin/β-catenin target gene downregulation (resembling MET), and co-immunoprecipitation showed Tspan8 and p120-catenin interact; Tspan8 mediates a several-fold increase in extracellular vesicle numbers in cell culture and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, EV quantification, Western blot, cell behavior assays\",\n      \"journal\": \"The Journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with partial mechanistic follow-up, single lab\",\n      \"pmids\": [\"30982971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"β-catenin stabilization occurs downstream of Tspan8 expression in melanoma; β-catenin in turn directly transcriptionally activates Tspan8, forming a positive feedback loop that sustains Tspan8-driven melanoma invasion.\",\n      \"method\": \"Tspan8 overexpression/knockdown, β-catenin reporter assay, ChIP, invasion assay, in vivo transgenic mouse model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP confirms direct transcriptional activation, feedback loop validated in vivo and in vitro, single lab\",\n      \"pmids\": [\"30679790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Tspan8+ melanoma cells cooperate with surrounding keratinocytes to activate keratinocyte-derived proMMP-9, degrade collagen IV, and achieve dermal colonization; this involves elevated MMP-3 and low TIMP-1 levels; anti-Tspan8 antibody reduces proMMP-9 activation and dermal invasion.\",\n      \"method\": \"3D skin reconstruct model, MMP activity assay, antibody blocking, siRNA knockdown\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional reconstitution in validated 3D model, multiple mechanistic readouts, antibody validation, single lab\",\n      \"pmids\": [\"32455575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EGFR signaling induces AKT-mediated phosphorylation of TSPAN8 at Ser129, enabling TSPAN8 binding to 14-3-3θ and importin-β1, which drives TSPAN8 nuclear translocation; in the nucleus, phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and transcription of MYC, BCL2, MMP9 and other oncogenes.\",\n      \"method\": \"Co-immunoprecipitation, phospho-site mutagenesis, nuclear fractionation, ChIP, reporter assays, in vitro kinase assay, antibody blocking in vivo\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis, ChIP, Co-IP, and in vivo validation with anti-TSPAN8 antibody, multiple orthogonal methods\",\n      \"pmids\": [\"35197608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TM4SF3/TSPAN8 physically interacts with AR-V7 (in addition to AR); interaction of TM4SF3 with AR or AR-V7 results in mutual deubiquitination and stabilization of both proteins; nuclear TM4SF3 is co-recruited to promoters of AR/AR-V7-regulated genes and is required for their transcriptional activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ChIP, nuclear fractionation, siRNA knockdown, domain mapping\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — Co-IP with ubiquitination assay, ChIP showing promoter co-recruitment, functional knockdown, multiple readouts\",\n      \"pmids\": [\"36951301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tspan8 coalesces with lipid rafts and facilitates IFN-γR1 localization at or near lipid rafts; Tspan8 silencing impairs lipid raft-mediated but promotes clathrin-mediated endocytosis of IFN-γR1, leading to increased STAT1 signaling and intestinal epithelial permeability.\",\n      \"method\": \"siRNA knockdown, lipid raft fractionation, endocytosis assay (clathrin vs lipid raft), STAT1 signaling readout, barrier permeability assay\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection of two endocytosis routes with functional consequence on signaling, single lab\",\n      \"pmids\": [\"37204469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tspan8 associates with endothelin-converting enzyme ECE1 and amplifies its enzymatic activity in converting big-ET1 to endothelin-1, demonstrated by transduction of Tspan8 into Isreco1 cells and by comparing ileum tissue of tspan8 knockout vs. wild-type mice.\",\n      \"method\": \"Mass spectrometry, Western blot co-immunoprecipitation, ECE1 activity assay, tspan8 knockout mouse tissue comparison\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified partner with enzymatic activity assay confirmed in KO mouse tissue, single lab\",\n      \"pmids\": [\"37835445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TSPAN8 promotes phosphorylation of E3 ligase RBBP6 at Ser772 by recruiting MAPK11, inducing SIRT6 protein degradation; SIRT6 downregulation subsequently upregulates GLS1 and PYCR1, causing TSPAN8+ myofibroblastic CAFs to secrete aspartate and proline that support breast cancer cell outgrowth.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, SIRT6 activity assay, metabolite secretion measurement, in vivo xenograft\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mechanistic Co-IP identifying kinase recruitment and substrate phosphorylation, downstream metabolic consequence validated in vivo\",\n      \"pmids\": [\"38569015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MDM2 acts as the common E3 ubiquitin ligase for TM4SF3/TSPAN8, AR, and AR-V7; MDM2 inhibition elevated all three proteins by reducing their ubiquitination; TM4SF3 interaction with AR or AR-V7 results in mutual deubiquitination and prevents proteasomal degradation.\",\n      \"method\": \"siRNA depletion of E3 ligases, MDM2 pharmacological inhibitor, ubiquitination assay, protein stability assay\",\n      \"journal\": \"Endocrine oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — identification of specific E3 ligase by systematic siRNA screen and pharmacological inhibition with ubiquitination readout, single lab\",\n      \"pmids\": [\"38410785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In Xenopus, tm4sf3 (TSPAN8 ortholog) is specifically expressed in the ventral pancreas at the fusion junction; morpholino-mediated knockdown inhibits dorsal-ventral pancreatic bud fusion and acinar cell differentiation, while overexpression promotes annular pancreas development.\",\n      \"method\": \"Morpholino knockdown, mRNA overexpression, Xenopus embryo model, in situ hybridization\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss- and gain-of-function in a vertebrate model with defined developmental phenotype, single lab\",\n      \"pmids\": [\"19403659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TSPAN8-high spermatogonia in mouse testis are enriched for spermatogonial stem cells (SSCs), as demonstrated by transplantation studies showing that TSPAN8-high subpopulations have greater stem cell activity than TSPAN8-low progenitor spermatogonia.\",\n      \"method\": \"Flow cytometry cell sorting, spermatogonial transplantation assay, RNA-seq, ChIP-seq, Methyl-seq\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transplantation (gold-standard SSC assay) directly links TSPAN8 expression to SSC identity, single lab\",\n      \"pmids\": [\"27733379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TSPAN8 promotes cancer cell stemness by activating Sonic Hedgehog signaling through exosomal transfer; CSC-derived TSPAN8-enriched exosomes activate Hh signaling in non-stem cancer cells, increasing their clonogenic ability, invasiveness, and chemoresistance in PDAC.\",\n      \"method\": \"Single-cell RNA sequencing, exosomal profiling, functional assays (clonogenicity, invasion, chemoresistance), in vivo tumor growth\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — exosome transfer experiments with pathway-specific functional readouts, in vivo validation, single lab\",\n      \"pmids\": [\"40251391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TSPAN8 directly interacts with β-catenin (by Co-IP), enhances β-catenin protein expression, and promotes colorectal cancer stemness; β-catenin in turn directly binds the TSPAN8 promoter (by ChIP) and enhances TSPAN8 transcription, forming a positive regulatory loop.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, siRNA knockdown, sphere formation assay, Western blot\",\n      \"journal\": \"Medical science monitor\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus ChIP establish bidirectional regulation with functional stemness readout, single lab\",\n      \"pmids\": [\"31838484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TSPAN8 forms a complex with Rictor (a component of mTORC2) and overexpression of TSPAN8 suppresses high-glucose-induced autophagy and apoptosis in kidney cells in an mTOR-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, TSPAN8 overexpression, mTOR inhibitor treatment, apoptosis/autophagy assays\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP identifying mTORC2 complex, functional assay without detailed mechanistic follow-up\",\n      \"pmids\": [\"35904232\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSPAN8 is a tetraspanin that functions as a membrane organizer forming complexes with integrins (α6β4, α6β1, α3β1), E-cadherin, EGFR, PTCH1, ECE1, AR, and mTORC2; it controls cell adhesion, migration, and invasion by regulating integrin-adhesion signaling and MMP activation, drives angiogenesis via selective loading of protein/mRNA cargo into exosomes bearing Tspan8-CD49d complexes, promotes cancer stemness by recruiting deubiquitinase ATXN3 to stabilize SHH/PTCH1 and activate Hedgehog signaling, undergoes EGFR-AKT-dependent phosphorylation at Ser129 enabling 14-3-3θ/importin-β1-mediated nuclear translocation where it co-activates STAT3 transcription, stabilizes AR/AR-V7 through mutual deubiquitination in prostate cancer, and regulates IFN-γR1 endocytosis route (lipid raft vs. clathrin) to control intestinal epithelial barrier integrity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TSPAN8 is a tetraspanin that organizes membrane microdomains to coordinate integrin-mediated adhesion, cell migration, extracellular vesicle biogenesis, and receptor signaling across epithelial, endothelial, and stromal compartments. At the plasma membrane, TSPAN8 forms complexes with integrins (α6β4, α6β1, α3β1), E-cadherin, and p120-catenin to regulate adhesion–motility switching through Src/FAK/Ras activation and MMP-dependent matrix remodeling, and it selectively loads cargo into exosomes that drive VEGF-independent angiogenesis and paracrine Hedgehog pathway activation in recipient cells [PMID:15837731, PMID:20858717, PMID:23683890, PMID:20124479, PMID:31253779]. TSPAN8 also undergoes EGFR–AKT-dependent phosphorylation at Ser129, enabling 14-3-3θ/importin-β1-mediated nuclear translocation where it co-activates STAT3 target genes, and in prostate cancer it stabilizes AR/AR-V7 through mutual protection from MDM2-mediated ubiquitination [PMID:35197608, PMID:26649804, PMID:36951301, PMID:38410785]. Beyond tumor biology, TSPAN8 directs IFN-γR1 into lipid-raft endocytic routes to restrain STAT1 signaling and maintain intestinal epithelial barrier integrity, interacts with ECE1 to amplify endothelin-1 production, and is required for ventral–dorsal pancreatic bud fusion during Xenopus development [PMID:37204469, PMID:37835445, PMID:19403659].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing TSPAN8 as a metastasis-associated tetraspanin that partners with integrins: transfection of D6.1A into a low-metastasizing line proved that TSPAN8 expression is sufficient to increase metastatic potential and demonstrated the first integrin partner (α6β1).\",\n      \"evidence\": \"cDNA transfection and Co-IP in rat tumor lines\",\n      \"pmids\": [\"9531564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reciprocal validation of integrin interaction with purified proteins\", \"Mechanism linking integrin association to metastasis not defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defining how TSPAN8–integrin complexes regulate cell behavior: PKC-induced strengthening of the TSPAN8–α6β4 complex caused integrin internalization, decreased laminin adhesion, and increased migration, linking tetraspanin–integrin dynamics to a motility switch.\",\n      \"evidence\": \"Co-IP, PKC activation, migration assay in human pancreatic carcinoma cells\",\n      \"pmids\": [\"15837731\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PKC substrate specificity on TSPAN8 or integrin not identified\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealing TSPAN8 as a driver of angiogenesis: tumor-cell and exosome-expressed TSPAN8 induced MMP/uPA secretion and VEGF/VEGFR upregulation, establishing a non-cell-autonomous pro-angiogenic role.\",\n      \"evidence\": \"In vivo angiogenesis assay with anti-D6.1A antibody blocking\",\n      \"pmids\": [\"16849554\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular mechanism linking TSPAN8 to VEGF induction unclear\", \"Exosome-specific versus cell-surface contribution not separated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating a developmental role for TSPAN8: morpholino knockdown in Xenopus showed TSPAN8 is required for dorsal–ventral pancreatic bud fusion and acinar differentiation, broadening its biology beyond cancer.\",\n      \"evidence\": \"Morpholino knockdown and mRNA overexpression in Xenopus embryos\",\n      \"pmids\": [\"19403659\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian pancreatic development role not confirmed\", \"Downstream signaling pathway mediating bud fusion unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Elucidating TSPAN8's role in exosome cargo selection and VEGF-independent angiogenesis: Tspan8–CD49d complexes selectively recruited proteins and mRNA into exosomes that were preferentially taken up by endothelial cells, activating angiogenic gene programs independently of VEGF.\",\n      \"evidence\": \"Exosome isolation, Co-IP, gene expression profiling, EC functional assays\",\n      \"pmids\": [\"20124479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sorting signal within TSPAN8 for exosome loading not mapped\", \"Whether TSPAN8 functions as cargo receptor or scaffold not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Dissecting TSPAN8 internalization and adhesion-complex remodeling: TSPAN8's N-terminal region recruits intersectin-2 into clathrin-dependent complexes with CD49d, explaining how activation-induced TSPAN8 endocytosis couples to reduced adhesion and enhanced migration, and how TSPAN8 interacts with E-cadherin and p120-catenin to switch between collagen-binding integrins α1β1 and α2β1.\",\n      \"evidence\": \"Chimera internalization assays, chemical cross-linking, Co-IP, siRNA knockdown, migration assays\",\n      \"pmids\": [\"20937409\", \"20858717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TSPAN8–E-cadherin interaction not resolved\", \"Whether intersectin-2 route and E-cadherin complex operate in the same cell context unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping TSPAN8 signaling downstream of integrin β4: TSPAN8 recruits β4 integrin, promotes its phosphorylation and Src/FAK/Ras activation, shifting cells from adhesion to motility, while CD151 preferentially associates with MMPs—establishing non-redundant tetraspanin functions.\",\n      \"evidence\": \"Stable knockdown, Co-IP, phosphorylation and signaling assays in rat pancreatic adenocarcinoma\",\n      \"pmids\": [\"23683890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for β4 phosphorylation not identified\", \"Whether TSPAN8 and CD151 compete for the same integrin pools unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovering TSPAN8 nuclear localization and AR stabilization: TSPAN8 was found in the nucleus of prostate cancer cells in an AR-dependent manner, directly interacting with AR and reducing its ubiquitin-proteasomal degradation, establishing TSPAN8 as a transcriptional co-regulator.\",\n      \"evidence\": \"Co-IP, nuclear fractionation, in vitro interaction assay, siRNA knockdown\",\n      \"pmids\": [\"26649804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deubiquitinase mediating AR stabilization by TSPAN8 not identified at this point\", \"Mechanism of TSPAN8 nuclear import not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"In vivo genetic validation: Tspan8/CD151 knockout mice showed impaired tumor dissemination with distorted tetraspanin–integrin–protease networks, and host-derived Tspan8-containing exosomes promoted angiogenesis through GPCR/RTK associations in endothelial cells.\",\n      \"evidence\": \"Knockout mice, tumor implantation, exosome coculture, Co-IP, rescue experiments\",\n      \"pmids\": [\"30541597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single vs. double KO contributions not fully deconvoluted\", \"Identity of specific GPCR/RTK partners on endothelial cells not determined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking TSPAN8 to Hedgehog pathway and cancer stemness: TSPAN8 recruited deubiquitinase ATXN3 to stabilize the SHH/PTCH1 complex, enabling SMO ciliary translocation and downstream Hh signaling that drove NANOG/OCT4 expression and chemoresistance; a β-catenin–TSPAN8 positive feedback loop was simultaneously identified in melanoma and colorectal cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, confocal cilia imaging, ChIP for β-catenin on TSPAN8 promoter, in vivo tumor formation\",\n      \"pmids\": [\"31253779\", \"30679790\", \"31838484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ATXN3 recruitment is direct or scaffold-mediated not resolved\", \"Intersection between Hh and β-catenin feedback loops not explored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defining the nuclear import mechanism: EGFR–AKT phosphorylates TSPAN8 at Ser129, creating a 14-3-3θ binding site that enables importin-β1-mediated nuclear translocation; nuclear phospho-TSPAN8 enhances STAT3 chromatin occupancy at MYC, BCL2, and MMP9 promoters.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mutagenesis, ChIP, nuclear fractionation, anti-TSPAN8 antibody in vivo\",\n      \"pmids\": [\"35197608\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether nuclear TSPAN8–STAT3 complex requires additional co-factors unknown\", \"Relationship between AR-dependent and EGFR-dependent nuclear import not clarified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extending the AR co-regulator role: TSPAN8 was shown to interact with AR-V7 in addition to full-length AR, with mutual deubiquitination enabling both proteins' stabilization and co-recruitment to AR/AR-V7-regulated gene promoters; separately, TSPAN8 was found to direct IFN-γR1 into lipid-raft endocytosis to restrain STAT1 signaling and maintain intestinal barrier integrity, and to complex with ECE1 to amplify endothelin-1 production.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, ChIP, lipid raft fractionation, ECE1 activity assay in Tspan8 KO mouse tissue\",\n      \"pmids\": [\"36951301\", \"37204469\", \"37835445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deubiquitinase responsible for AR/TSPAN8 mutual stabilization still unidentified\", \"Whether ECE1 activation is direct or via membrane microdomain organization not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealing a stromal metabolic reprogramming function: in myofibroblastic cancer-associated fibroblasts, TSPAN8 recruits MAPK11 to phosphorylate E3 ligase RBBP6 at Ser772, triggering SIRT6 degradation and upregulating GLS1/PYCR1-dependent aspartate and proline secretion that feeds tumor cell outgrowth; MDM2 was identified as the E3 ligase mediating ubiquitination of TSPAN8, AR, and AR-V7.\",\n      \"evidence\": \"Co-IP, phosphorylation assay, metabolite measurement, in vivo xenograft; systematic E3 ligase siRNA screen with MDM2 inhibitor\",\n      \"pmids\": [\"38569015\", \"38410785\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MAPK11 recruitment is a general TSPAN8 function or CAF-specific unknown\", \"Structural basis of TSPAN8–MDM2 interaction not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis of TSPAN8's multivalent scaffolding across integrins, receptors, and nuclear partners; whether the EGFR–AKT and AR-dependent nuclear import pathways converge or operate independently; the identity of the deubiquitinase(s) mediating mutual AR/TSPAN8 stabilization; and how TSPAN8 cargo-sorting specificity in exosomes is encoded.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of TSPAN8 in complex with any partner\", \"No reconstitution of TSPAN8-dependent exosome sorting with purified components\", \"Nuclear versus membrane pool regulation not mechanistically separated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4, 5, 12, 16, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12, 18, 19]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [9, 16, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 5, 18]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 16, 17]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 8, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [5, 6, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 16, 18, 25]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 3, 7, 8, 11, 15, 20]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 8, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [7, 15]}\n    ],\n    \"complexes\": [\n      \"TSPAN8–integrin α6β4 complex\",\n      \"TSPAN8–E-cadherin–p120-catenin complex\",\n      \"TSPAN8–AR/AR-V7 complex\",\n      \"TSPAN8–PTCH1–ATXN3 complex\"\n    ],\n    \"partners\": [\n      \"ITGB4\",\n      \"ITGA6\",\n      \"CDH1\",\n      \"CTNND1\",\n      \"AR\",\n      \"PTCH1\",\n      \"ATXN3\",\n      \"STAT3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}