{"gene":"HOXA6","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1994,"finding":"Targeted disruption of murine Hoxa-6 causes homeotic transformation of the seventh cervical vertebra to the first thoracic vertebra, demonstrating that Hoxa-6 is required to specify vertebral identity at its anterior expression boundary.","method":"Gene targeting (knockout mouse), skeletal phenotype analysis","journal":"Mechanisms of development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function knockout with specific skeletal phenotype, replicated across multiple animals; foundational developmental genetics study","pmids":["7918106"],"is_preprint":false},{"year":2009,"finding":"Enforced overexpression of HOXA6 in FDCP-Mix multipotential hemopoietic progenitor cells increases proliferation and colony formation, and potentiates factor-independent proliferation in both FDCP-Mix and Ba/F3 cells, demonstrating a direct role for HOXA6 in hemopoietic progenitor self-renewal and proliferation.","method":"Retroviral overexpression in hemopoietic cell lines, colony formation assay, factor-independent proliferation assay","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain-of-function in multiple cell lines with defined cellular phenotype, single lab, no rescue or epistasis","pmids":["19157684"],"is_preprint":false},{"year":2018,"finding":"In colorectal cancer cells, HOXA6 overexpression promotes cell proliferation, migration, and invasion while inhibiting apoptosis; it suppresses Bax, caspase-3, and PARP expression and E-cadherin while upregulating Bcl-2, N-cadherin, and Vimentin, indicating HOXA6 regulates apoptosis via the Bcl-2 pathway and promotes invasion through EMT.","method":"Plasmid overexpression and siRNA knockdown in CRC cell lines, CCK-8, colony formation, EdU, TUNEL, flow cytometry, Transwell, wound-healing, Western blot","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — bidirectional manipulation (OE and KD) with multiple orthogonal readouts in two cell lines, single lab","pmids":["29620285"],"is_preprint":false},{"year":2019,"finding":"In clear cell renal cell carcinoma cells, HOXA6 overexpression inhibits cell proliferation and promotes apoptosis by suppressing PI3K, phospho-Akt, MEK, phospho-ERK, and Bcl-2, while increasing PTEN, Bax, cleaved caspase-3, and cleaved PARP levels, placing HOXA6 as a negative regulator of the PI3K/Akt/ERK cascade.","method":"Plasmid overexpression and shRNA knockdown in 786-O and 769-P cell lines, CCK-8, colony formation, EdU, Caspase-Glo, TUNEL, flow cytometry, Western blot","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — bidirectional manipulation with multiple functional readouts and Western blot pathway analysis, single lab","pmids":["31081053"],"is_preprint":false},{"year":2020,"finding":"In rat spinal dorsal horn, oxaliplatin induces TET1-mediated demethylation of the SOX10 promoter, which upregulates SOX10; SOX10 then binds directly to the HOXA6 promoter (confirmed by ChIP) and increases HOXA6 expression, contributing to mechanical allodynia. siRNA knockdown of HOXA6 alleviated allodynia, placing HOXA6 downstream of the TET1–SOX10 axis in oxaliplatin-induced neuropathic pain.","method":"Whole-genome expression microarray, RRBS (reduced representation bisulfite sequencing), siRNA knockdown (HOXA6, TET1, SOX10), AAV-SOX10 overexpression, chromatin immunoprecipitation (ChIP), behavioral assays (mechanical allodynia)","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP confirms direct SOX10 binding to HOXA6 promoter; multiple orthogonal methods (RRBS, ChIP, siRNA, AAV); single lab","pmids":["32428246"],"is_preprint":false},{"year":2021,"finding":"HOXA6 physically interacts with PBX2 and stabilizes the PBX2 protein in gastric cancer cells; co-overexpression enhances migration and invasion more than either alone, and in vivo orthotopic implantation confirms cooperative enhancement of metastasis.","method":"Co-immunoprecipitation (physical interaction), siRNA knockdown, overexpression, Transwell migration/invasion assays, orthotopic mouse model, TMA/IHC","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — physical interaction shown by Co-IP, functional cooperation validated in vitro and in vivo, single lab","pmids":["33535170"],"is_preprint":false},{"year":2023,"finding":"HOXA6 acts as a transcriptional activator of ZBTB12 in cancer-associated fibroblasts: HOXA6 binds the ZBTB12 promoter (confirmed by ChIP in 293T cells and CAFs), and HOXA6 silencing reduces ZBTB12 mRNA and protein; this HOXA6/ZBTB12 axis mediates the pro-tumorigenic effects of CAFs on gastric cancer cells.","method":"ChIP assay (HOXA6 binding to ZBTB12 promoter), siRNA knockdown, overexpression, proliferation/migration/invasion assays in co-culture, Western blot, bioinformatics (hTFtarget)","journal":"Acta pharmaceutica (Zagreb, Croatia)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter binding confirmed by ChIP in two cell types with functional rescue, single lab","pmids":["37708965"],"is_preprint":false},{"year":2025,"finding":"In non-small-cell lung cancer, KIAA1429 (VIRMA) enhances lncRNA TRERNA1 stability via m6A modification; TRERNA1 recruits EZH2 to the HOXA6 promoter, increasing H3K27me3 and suppressing HOXA6 expression. HOXA6 downregulation is required for the pro-proliferative effect of KIAA1429, as HOXA6 re-expression partially rescues growth inhibition caused by KIAA1429 knockdown.","method":"m6A methylation assay, RNA stability assay, EZH2 ChIP (H3K27me3 at HOXA6 promoter), siRNA/shRNA knockdown, overexpression, proliferation assays, in vivo xenograft","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP confirms EZH2/H3K27me3 at HOXA6 promoter; multiple orthogonal methods with in vivo validation; single lab","pmids":["40640307"],"is_preprint":false},{"year":2025,"finding":"DDR1 inhibits ferroptosis in bladder cancer cells by upregulating HOXA6; DDR1 knockdown-induced ferroptotic cell death (decreased glutathione, GPX4, SLC7A11; increased MDA and Fe2+) is abrogated by HOXA6 knockdown, placing HOXA6 downstream of DDR1 in the regulation of ferroptosis.","method":"siRNA knockdown of DDR1 and HOXA6, ferroptosis inducers/inhibitors (erastin, ferrostatin-1), biochemical assays (GSH, GPX4, SLC7A11, MDA, Fe2+ levels, ACSL4, EMT markers), in vivo xenograft","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — epistasis shown by double knockdown with multiple biochemical readouts; single lab, no reconstitution","pmids":["40105492"],"is_preprint":false}],"current_model":"HOXA6 is a homeodomain transcription factor that specifies vertebral identity during embryogenesis (loss-of-function causes cervical-to-thoracic homeotic transformation), promotes hemopoietic progenitor self-renewal, physically interacts with PBX2 to enhance cancer cell invasiveness, directly binds the ZBTB12 promoter to drive its transcription, suppresses the PI3K/Akt/ERK pathway to limit tumor cell proliferation in some contexts, acts downstream of a TET1–SOX10 epigenetic axis to contribute to neuropathic pain, and is itself transcriptionally silenced by EZH2-mediated H3K27me3 deposition recruited via lncRNA TRERNA1."},"narrative":{"mechanistic_narrative":"HOXA6 is a homeodomain transcription factor that specifies axial vertebral identity during embryogenesis, where targeted disruption in mouse causes a homeotic transformation of the seventh cervical vertebra to the first thoracic vertebra at its anterior expression boundary [PMID:7918106]. Beyond development, HOXA6 regulates progenitor proliferation and self-renewal, as its enforced overexpression drives proliferation, colony formation, and factor-independent growth in multipotential hemopoietic progenitor cells [PMID:19157684]. As a transcriptional regulator it acts directly on target promoters: HOXA6 binds the ZBTB12 promoter to activate its transcription, an axis that mediates the pro-tumorigenic effects of cancer-associated fibroblasts on gastric cancer cells [PMID:37708965]. It also functions through protein partnership, physically interacting with and stabilizing PBX2 to cooperatively enhance gastric cancer migration, invasion, and metastasis [PMID:33535170]. In cancer contexts HOXA6 shows context-dependent roles, promoting proliferation, EMT, and survival in colorectal cancer via Bcl-2 pathway regulation [PMID:29620285], yet acting as a negative regulator of the PI3K/Akt/ERK cascade to inhibit proliferation and promote apoptosis in clear cell renal cell carcinoma [PMID:31081053]. HOXA6 is positioned within several regulatory circuits as both an effector and a target: it operates downstream of a TET1–SOX10 epigenetic axis to contribute to oxaliplatin-induced neuropathic pain [PMID:32428246], downstream of DDR1 to suppress ferroptosis in bladder cancer [PMID:40105492], and is itself transcriptionally silenced through EZH2-mediated H3K27me3 deposition recruited by the m6A-stabilized lncRNA TRERNA1 [PMID:40640307].","teleology":[{"year":1994,"claim":"Established HOXA6's foundational in vivo function by asking what role it plays in axial patterning; the knockout revealed it is required to specify vertebral identity at its anterior expression boundary.","evidence":"Targeted gene disruption in mouse with skeletal phenotype analysis","pmids":["7918106"],"confidence":"High","gaps":["Does not identify direct downstream transcriptional targets in the developmental context","No molecular mechanism (DNA binding sites, cofactors) defined in vivo"]},{"year":2009,"claim":"Extended HOXA6 function beyond development by testing its effect on blood progenitors; overexpression showed it can drive self-renewal and factor-independent proliferation.","evidence":"Retroviral overexpression in FDCP-Mix and Ba/F3 hemopoietic cell lines with colony and proliferation assays","pmids":["19157684"],"confidence":"Medium","gaps":["Gain-of-function only; no loss-of-function or rescue","Downstream effectors not identified","Single lab"]},{"year":2018,"claim":"Addressed whether HOXA6 contributes to tumor progression; bidirectional manipulation in colorectal cancer showed it promotes proliferation, invasion, and EMT while suppressing apoptosis via the Bcl-2 pathway.","evidence":"Overexpression and siRNA knockdown in CRC cell lines with multiple orthogonal functional and Western blot readouts","pmids":["29620285"],"confidence":"Medium","gaps":["Direct transcriptional targets driving these phenotypes not identified","No in vivo validation","Single lab"]},{"year":2019,"claim":"Tested HOXA6's role in a different tumor type and revealed a context-dependent, opposite function: it inhibits proliferation and promotes apoptosis by negatively regulating the PI3K/Akt/ERK cascade in renal carcinoma.","evidence":"Overexpression and shRNA knockdown in 786-O and 769-P cells with functional assays and pathway Western blots","pmids":["31081053"],"confidence":"Medium","gaps":["Mechanism by which HOXA6 represses the pathway (direct vs indirect) not defined","Reconciliation with pro-tumor roles in other cancers unresolved","Single lab"]},{"year":2020,"claim":"Defined an upstream regulatory input by asking how HOXA6 is induced in neuropathic pain; ChIP showed SOX10 binds the HOXA6 promoter, placing it downstream of a TET1–SOX10 epigenetic axis driving allodynia.","evidence":"RRBS, ChIP, siRNA knockdown, AAV overexpression, and behavioral assays in rat spinal dorsal horn","pmids":["32428246"],"confidence":"Medium","gaps":["HOXA6 transcriptional targets in nociceptive neurons not identified","Single lab"]},{"year":2021,"claim":"Identified a direct protein partner by asking how HOXA6 promotes metastasis; Co-IP showed it binds and stabilizes PBX2, with cooperative enhancement of invasion and metastasis.","evidence":"Co-immunoprecipitation, knockdown/overexpression, Transwell assays, and orthotopic mouse model in gastric cancer","pmids":["33535170"],"confidence":"Medium","gaps":["Reciprocal interaction validation and mapping of binding interface not done","Mechanism of PBX2 stabilization unclear","Single lab"]},{"year":2023,"claim":"Defined a direct transcriptional target by asking what genes HOXA6 activates; ChIP confirmed binding to the ZBTB12 promoter, establishing a HOXA6/ZBTB12 axis mediating CAF pro-tumorigenic effects.","evidence":"ChIP in 293T and CAFs, knockdown/overexpression, co-culture functional assays, and Western blot","pmids":["37708965"],"confidence":"Medium","gaps":["Genome-wide target repertoire not defined","Whether ZBTB12 fully accounts for the phenotype unresolved","Single lab"]},{"year":2025,"claim":"Established how HOXA6 itself is silenced in lung cancer; an m6A-stabilized lncRNA TRERNA1 recruits EZH2 to deposit H3K27me3 at the HOXA6 promoter, and HOXA6 re-expression rescues growth inhibition.","evidence":"m6A and RNA stability assays, EZH2/H3K27me3 ChIP, knockdown/overexpression, and xenograft in NSCLC","pmids":["40640307"],"confidence":"Medium","gaps":["Direct HOXA6 targets mediating growth suppression in NSCLC not identified","Single lab"]},{"year":2025,"claim":"Placed HOXA6 in a ferroptosis-regulatory circuit by asking how DDR1 protects bladder cancer cells; double-knockdown epistasis showed HOXA6 acts downstream of DDR1 to suppress ferroptotic death.","evidence":"siRNA double knockdown, ferroptosis inducers/inhibitors, biochemical ferroptosis markers, and xenograft in bladder cancer","pmids":["40105492"],"confidence":"Medium","gaps":["Direct HOXA6 transcriptional targets controlling ferroptosis genes (GPX4, SLC7A11) not defined","No reconstitution","Single lab"]},{"year":null,"claim":"The unifying molecular basis of HOXA6's context-dependent and opposite roles across tumor types, and its direct genome-wide DNA-binding target repertoire, remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No genome-wide HOXA6 ChIP-seq target map in the corpus","Mechanism distinguishing pro- vs anti-proliferative outcomes unresolved","No structural model of HOXA6–DNA or HOXA6–PBX2 interaction"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,4,6]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[6,4]}],"complexes":[],"partners":["PBX2","ZBTB12","SOX10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P31267","full_name":"Homeobox protein Hox-A6","aliases":["Homeobox protein Hox-1B"],"length_aa":233,"mass_kda":26.3,"function":"Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P31267/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HOXA6","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HOXA6","total_profiled":1310},"omim":[{"mim_id":"609907","title":"SEMAPHORIN 3D; SEMA3D","url":"https://www.omim.org/entry/609907"},{"mim_id":"142958","title":"HOMEOBOX A11; HOXA11","url":"https://www.omim.org/entry/142958"},{"mim_id":"142953","title":"HOMEOBOX A4; HOXA4","url":"https://www.omim.org/entry/142953"},{"mim_id":"142951","title":"HOMEOBOX A6; HOXA6","url":"https://www.omim.org/entry/142951"},{"mim_id":"142950","title":"HOMEOBOX A7; HOXA7","url":"https://www.omim.org/entry/142950"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"fallopian tube","ntpm":11.2}],"url":"https://www.proteinatlas.org/search/HOXA6"},"hgnc":{"alias_symbol":[],"prev_symbol":["HOX1B","HOX1"]},"alphafold":{"accession":"P31267","domains":[{"cath_id":"1.10.10.60","chopping":"163-217","consensus_level":"medium","plddt":96.4024,"start":163,"end":217}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P31267","model_url":"https://alphafold.ebi.ac.uk/files/AF-P31267-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P31267-F1-predicted_aligned_error_v6.png","plddt_mean":62.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HOXA6","jax_strain_url":"https://www.jax.org/strain/search?query=HOXA6"},"sequence":{"accession":"P31267","fasta_url":"https://rest.uniprot.org/uniprotkb/P31267.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P31267/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P31267"}},"corpus_meta":[{"pmid":"21925392","id":"PMC_21925392","title":"The noncoding RNA Mistral activates Hoxa6 and Hoxa7 expression and stem cell differentiation by recruiting MLL1 to chromatin.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21925392","citation_count":185,"is_preprint":false},{"pmid":"7918106","id":"PMC_7918106","title":"Targeted disruptions of the murine Hoxa-4 and Hoxa-6 genes result in homeotic transformations of components of the vertebral column.","date":"1994","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/7918106","citation_count":102,"is_preprint":false},{"pmid":"31165042","id":"PMC_31165042","title":"Study of Promoter Methylation Patterns of HOXA2, HOXA5, and HOXA6 and Its Clinicopathological Characteristics in Colorectal Cancer.","date":"2019","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/31165042","citation_count":35,"is_preprint":false},{"pmid":"31081053","id":"PMC_31081053","title":"HOXA6 inhibits cell proliferation and induces apoptosis by suppressing the PI3K/Akt signaling pathway in clear cell renal cell carcinoma.","date":"2019","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/31081053","citation_count":21,"is_preprint":false},{"pmid":"19157684","id":"PMC_19157684","title":"Hoxa6 potentiates short-term hemopoietic cell proliferation and extended self-renewal.","date":"2009","source":"Experimental hematology","url":"https://pubmed.ncbi.nlm.nih.gov/19157684","citation_count":20,"is_preprint":false},{"pmid":"31114997","id":"PMC_31114997","title":"MiR-1294 acts as a tumor suppressor in clear cell renal cell carcinoma through targeting HOXA6.","date":"2019","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31114997","citation_count":19,"is_preprint":false},{"pmid":"29620285","id":"PMC_29620285","title":"Effect of HOXA6 on the proliferation, apoptosis, migration and invasion of colorectal cancer cells.","date":"2018","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/29620285","citation_count":19,"is_preprint":false},{"pmid":"33535170","id":"PMC_33535170","title":"Coexpression of HOXA6 and PBX2 promotes metastasis in gastric cancer.","date":"2021","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/33535170","citation_count":16,"is_preprint":false},{"pmid":"32428246","id":"PMC_32428246","title":"Oxaliplatin-induced neuropathic pain involves HOXA6 via a TET1-dependent demethylation of the SOX10 promoter.","date":"2020","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/32428246","citation_count":15,"is_preprint":false},{"pmid":"35349479","id":"PMC_35349479","title":"Next-generation sequencing identifies HOXA6 as a novel oncogenic gene in low grade glioma.","date":"2022","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/35349479","citation_count":13,"is_preprint":false},{"pmid":"37708965","id":"PMC_37708965","title":"Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts through regulation of the HOXA6/ZBTB12 axis.","date":"2023","source":"Acta pharmaceutica (Zagreb, Croatia)","url":"https://pubmed.ncbi.nlm.nih.gov/37708965","citation_count":7,"is_preprint":false},{"pmid":"31159758","id":"PMC_31159758","title":"A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth.","date":"2019","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31159758","citation_count":5,"is_preprint":false},{"pmid":"40640307","id":"PMC_40640307","title":"KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40640307","citation_count":2,"is_preprint":false},{"pmid":"40105492","id":"PMC_40105492","title":"DDR1 Targeting HOXA6 Facilitates Bladder Cancer Progression via Inhibiting Ferroptosis.","date":"2025","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40105492","citation_count":1,"is_preprint":false},{"pmid":"39345473","id":"PMC_39345473","title":"Single Cell Profiling in the Sox10  Hirschsprung Mouse Implicates Hoxa6 in Enteric Neuron Lineage Allocation.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39345473","citation_count":0,"is_preprint":false},{"pmid":"38884519","id":"PMC_38884519","title":"Retraction Note: MiR-1294 acts as a tumor suppressor in clear cell renal cell carcinoma through targeting HOXA6.","date":"2024","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/38884519","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.09.18.613729","title":"Single Cell Profiling in the  <i>  Sox10  <sup>Dom/+</sup>  </i>  Hirschsprung Mouse Implicates  <i>Hoxa6</i>  in Enteric Neuron Lineage Allocation","date":"2024-09-19","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.18.613729","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11956,"output_tokens":2533,"usd":0.036931,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9748,"output_tokens":3261,"usd":0.065132,"stage2_stop_reason":"end_turn"},"total_usd":0.102063,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"Targeted disruption of murine Hoxa-6 causes homeotic transformation of the seventh cervical vertebra to the first thoracic vertebra, demonstrating that Hoxa-6 is required to specify vertebral identity at its anterior expression boundary.\",\n      \"method\": \"Gene targeting (knockout mouse), skeletal phenotype analysis\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function knockout with specific skeletal phenotype, replicated across multiple animals; foundational developmental genetics study\",\n      \"pmids\": [\"7918106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Enforced overexpression of HOXA6 in FDCP-Mix multipotential hemopoietic progenitor cells increases proliferation and colony formation, and potentiates factor-independent proliferation in both FDCP-Mix and Ba/F3 cells, demonstrating a direct role for HOXA6 in hemopoietic progenitor self-renewal and proliferation.\",\n      \"method\": \"Retroviral overexpression in hemopoietic cell lines, colony formation assay, factor-independent proliferation assay\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain-of-function in multiple cell lines with defined cellular phenotype, single lab, no rescue or epistasis\",\n      \"pmids\": [\"19157684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In colorectal cancer cells, HOXA6 overexpression promotes cell proliferation, migration, and invasion while inhibiting apoptosis; it suppresses Bax, caspase-3, and PARP expression and E-cadherin while upregulating Bcl-2, N-cadherin, and Vimentin, indicating HOXA6 regulates apoptosis via the Bcl-2 pathway and promotes invasion through EMT.\",\n      \"method\": \"Plasmid overexpression and siRNA knockdown in CRC cell lines, CCK-8, colony formation, EdU, TUNEL, flow cytometry, Transwell, wound-healing, Western blot\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — bidirectional manipulation (OE and KD) with multiple orthogonal readouts in two cell lines, single lab\",\n      \"pmids\": [\"29620285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In clear cell renal cell carcinoma cells, HOXA6 overexpression inhibits cell proliferation and promotes apoptosis by suppressing PI3K, phospho-Akt, MEK, phospho-ERK, and Bcl-2, while increasing PTEN, Bax, cleaved caspase-3, and cleaved PARP levels, placing HOXA6 as a negative regulator of the PI3K/Akt/ERK cascade.\",\n      \"method\": \"Plasmid overexpression and shRNA knockdown in 786-O and 769-P cell lines, CCK-8, colony formation, EdU, Caspase-Glo, TUNEL, flow cytometry, Western blot\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — bidirectional manipulation with multiple functional readouts and Western blot pathway analysis, single lab\",\n      \"pmids\": [\"31081053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In rat spinal dorsal horn, oxaliplatin induces TET1-mediated demethylation of the SOX10 promoter, which upregulates SOX10; SOX10 then binds directly to the HOXA6 promoter (confirmed by ChIP) and increases HOXA6 expression, contributing to mechanical allodynia. siRNA knockdown of HOXA6 alleviated allodynia, placing HOXA6 downstream of the TET1–SOX10 axis in oxaliplatin-induced neuropathic pain.\",\n      \"method\": \"Whole-genome expression microarray, RRBS (reduced representation bisulfite sequencing), siRNA knockdown (HOXA6, TET1, SOX10), AAV-SOX10 overexpression, chromatin immunoprecipitation (ChIP), behavioral assays (mechanical allodynia)\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP confirms direct SOX10 binding to HOXA6 promoter; multiple orthogonal methods (RRBS, ChIP, siRNA, AAV); single lab\",\n      \"pmids\": [\"32428246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOXA6 physically interacts with PBX2 and stabilizes the PBX2 protein in gastric cancer cells; co-overexpression enhances migration and invasion more than either alone, and in vivo orthotopic implantation confirms cooperative enhancement of metastasis.\",\n      \"method\": \"Co-immunoprecipitation (physical interaction), siRNA knockdown, overexpression, Transwell migration/invasion assays, orthotopic mouse model, TMA/IHC\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — physical interaction shown by Co-IP, functional cooperation validated in vitro and in vivo, single lab\",\n      \"pmids\": [\"33535170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HOXA6 acts as a transcriptional activator of ZBTB12 in cancer-associated fibroblasts: HOXA6 binds the ZBTB12 promoter (confirmed by ChIP in 293T cells and CAFs), and HOXA6 silencing reduces ZBTB12 mRNA and protein; this HOXA6/ZBTB12 axis mediates the pro-tumorigenic effects of CAFs on gastric cancer cells.\",\n      \"method\": \"ChIP assay (HOXA6 binding to ZBTB12 promoter), siRNA knockdown, overexpression, proliferation/migration/invasion assays in co-culture, Western blot, bioinformatics (hTFtarget)\",\n      \"journal\": \"Acta pharmaceutica (Zagreb, Croatia)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter binding confirmed by ChIP in two cell types with functional rescue, single lab\",\n      \"pmids\": [\"37708965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In non-small-cell lung cancer, KIAA1429 (VIRMA) enhances lncRNA TRERNA1 stability via m6A modification; TRERNA1 recruits EZH2 to the HOXA6 promoter, increasing H3K27me3 and suppressing HOXA6 expression. HOXA6 downregulation is required for the pro-proliferative effect of KIAA1429, as HOXA6 re-expression partially rescues growth inhibition caused by KIAA1429 knockdown.\",\n      \"method\": \"m6A methylation assay, RNA stability assay, EZH2 ChIP (H3K27me3 at HOXA6 promoter), siRNA/shRNA knockdown, overexpression, proliferation assays, in vivo xenograft\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP confirms EZH2/H3K27me3 at HOXA6 promoter; multiple orthogonal methods with in vivo validation; single lab\",\n      \"pmids\": [\"40640307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DDR1 inhibits ferroptosis in bladder cancer cells by upregulating HOXA6; DDR1 knockdown-induced ferroptotic cell death (decreased glutathione, GPX4, SLC7A11; increased MDA and Fe2+) is abrogated by HOXA6 knockdown, placing HOXA6 downstream of DDR1 in the regulation of ferroptosis.\",\n      \"method\": \"siRNA knockdown of DDR1 and HOXA6, ferroptosis inducers/inhibitors (erastin, ferrostatin-1), biochemical assays (GSH, GPX4, SLC7A11, MDA, Fe2+ levels, ACSL4, EMT markers), in vivo xenograft\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — epistasis shown by double knockdown with multiple biochemical readouts; single lab, no reconstitution\",\n      \"pmids\": [\"40105492\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HOXA6 is a homeodomain transcription factor that specifies vertebral identity during embryogenesis (loss-of-function causes cervical-to-thoracic homeotic transformation), promotes hemopoietic progenitor self-renewal, physically interacts with PBX2 to enhance cancer cell invasiveness, directly binds the ZBTB12 promoter to drive its transcription, suppresses the PI3K/Akt/ERK pathway to limit tumor cell proliferation in some contexts, acts downstream of a TET1–SOX10 epigenetic axis to contribute to neuropathic pain, and is itself transcriptionally silenced by EZH2-mediated H3K27me3 deposition recruited via lncRNA TRERNA1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HOXA6 is a homeodomain transcription factor that specifies axial vertebral identity during embryogenesis, where targeted disruption in mouse causes a homeotic transformation of the seventh cervical vertebra to the first thoracic vertebra at its anterior expression boundary [#0]. Beyond development, HOXA6 regulates progenitor proliferation and self-renewal, as its enforced overexpression drives proliferation, colony formation, and factor-independent growth in multipotential hemopoietic progenitor cells [#1]. As a transcriptional regulator it acts directly on target promoters: HOXA6 binds the ZBTB12 promoter to activate its transcription, an axis that mediates the pro-tumorigenic effects of cancer-associated fibroblasts on gastric cancer cells [#6]. It also functions through protein partnership, physically interacting with and stabilizing PBX2 to cooperatively enhance gastric cancer migration, invasion, and metastasis [#5]. In cancer contexts HOXA6 shows context-dependent roles, promoting proliferation, EMT, and survival in colorectal cancer via Bcl-2 pathway regulation [#2], yet acting as a negative regulator of the PI3K/Akt/ERK cascade to inhibit proliferation and promote apoptosis in clear cell renal cell carcinoma [#3]. HOXA6 is positioned within several regulatory circuits as both an effector and a target: it operates downstream of a TET1–SOX10 epigenetic axis to contribute to oxaliplatin-induced neuropathic pain [#4], downstream of DDR1 to suppress ferroptosis in bladder cancer [#8], and is itself transcriptionally silenced through EZH2-mediated H3K27me3 deposition recruited by the m6A-stabilized lncRNA TRERNA1 [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established HOXA6's foundational in vivo function by asking what role it plays in axial patterning; the knockout revealed it is required to specify vertebral identity at its anterior expression boundary.\",\n      \"evidence\": \"Targeted gene disruption in mouse with skeletal phenotype analysis\",\n      \"pmids\": [\"7918106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify direct downstream transcriptional targets in the developmental context\", \"No molecular mechanism (DNA binding sites, cofactors) defined in vivo\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended HOXA6 function beyond development by testing its effect on blood progenitors; overexpression showed it can drive self-renewal and factor-independent proliferation.\",\n      \"evidence\": \"Retroviral overexpression in FDCP-Mix and Ba/F3 hemopoietic cell lines with colony and proliferation assays\",\n      \"pmids\": [\"19157684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Gain-of-function only; no loss-of-function or rescue\", \"Downstream effectors not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Addressed whether HOXA6 contributes to tumor progression; bidirectional manipulation in colorectal cancer showed it promotes proliferation, invasion, and EMT while suppressing apoptosis via the Bcl-2 pathway.\",\n      \"evidence\": \"Overexpression and siRNA knockdown in CRC cell lines with multiple orthogonal functional and Western blot readouts\",\n      \"pmids\": [\"29620285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets driving these phenotypes not identified\", \"No in vivo validation\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Tested HOXA6's role in a different tumor type and revealed a context-dependent, opposite function: it inhibits proliferation and promotes apoptosis by negatively regulating the PI3K/Akt/ERK cascade in renal carcinoma.\",\n      \"evidence\": \"Overexpression and shRNA knockdown in 786-O and 769-P cells with functional assays and pathway Western blots\",\n      \"pmids\": [\"31081053\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which HOXA6 represses the pathway (direct vs indirect) not defined\", \"Reconciliation with pro-tumor roles in other cancers unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined an upstream regulatory input by asking how HOXA6 is induced in neuropathic pain; ChIP showed SOX10 binds the HOXA6 promoter, placing it downstream of a TET1–SOX10 epigenetic axis driving allodynia.\",\n      \"evidence\": \"RRBS, ChIP, siRNA knockdown, AAV overexpression, and behavioral assays in rat spinal dorsal horn\",\n      \"pmids\": [\"32428246\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HOXA6 transcriptional targets in nociceptive neurons not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a direct protein partner by asking how HOXA6 promotes metastasis; Co-IP showed it binds and stabilizes PBX2, with cooperative enhancement of invasion and metastasis.\",\n      \"evidence\": \"Co-immunoprecipitation, knockdown/overexpression, Transwell assays, and orthotopic mouse model in gastric cancer\",\n      \"pmids\": [\"33535170\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal interaction validation and mapping of binding interface not done\", \"Mechanism of PBX2 stabilization unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined a direct transcriptional target by asking what genes HOXA6 activates; ChIP confirmed binding to the ZBTB12 promoter, establishing a HOXA6/ZBTB12 axis mediating CAF pro-tumorigenic effects.\",\n      \"evidence\": \"ChIP in 293T and CAFs, knockdown/overexpression, co-culture functional assays, and Western blot\",\n      \"pmids\": [\"37708965\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genome-wide target repertoire not defined\", \"Whether ZBTB12 fully accounts for the phenotype unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established how HOXA6 itself is silenced in lung cancer; an m6A-stabilized lncRNA TRERNA1 recruits EZH2 to deposit H3K27me3 at the HOXA6 promoter, and HOXA6 re-expression rescues growth inhibition.\",\n      \"evidence\": \"m6A and RNA stability assays, EZH2/H3K27me3 ChIP, knockdown/overexpression, and xenograft in NSCLC\",\n      \"pmids\": [\"40640307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct HOXA6 targets mediating growth suppression in NSCLC not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed HOXA6 in a ferroptosis-regulatory circuit by asking how DDR1 protects bladder cancer cells; double-knockdown epistasis showed HOXA6 acts downstream of DDR1 to suppress ferroptotic death.\",\n      \"evidence\": \"siRNA double knockdown, ferroptosis inducers/inhibitors, biochemical ferroptosis markers, and xenograft in bladder cancer\",\n      \"pmids\": [\"40105492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct HOXA6 transcriptional targets controlling ferroptosis genes (GPX4, SLC7A11) not defined\", \"No reconstitution\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The unifying molecular basis of HOXA6's context-dependent and opposite roles across tumor types, and its direct genome-wide DNA-binding target repertoire, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No genome-wide HOXA6 ChIP-seq target map in the corpus\", \"Mechanism distinguishing pro- vs anti-proliferative outcomes unresolved\", \"No structural model of HOXA6–DNA or HOXA6–PBX2 interaction\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [6, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PBX2\", \"ZBTB12\", \"SOX10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}