{"gene":"CST6","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2021,"finding":"CST6 (cystatin E/M) inhibits osteolytic bone metastasis by entering osteoclasts via endocytosis and suppressing the cysteine protease CTSB (cathepsin B), leading to upregulation of the CTSB hydrolytic substrate SPHK1, which in turn suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, establishing a CST6-CTSB-SPHK1 signaling axis in osteoclast differentiation.","method":"In vitro osteoclastogenesis assay, in vivo metastasis assay, endocytosis tracking, recombinant protein treatment, peptide functional screening, preclinical metastasis models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal in vitro and in vivo methods, mechanistic pathway dissected with recombinant protein and functional assays, replicated in preclinical models","pmids":["34815788"],"is_preprint":false},{"year":2022,"finding":"CST6 suppresses osteolytic bone disease in multiple myeloma by inhibiting cathepsin K activity and blocking osteoclast differentiation; mechanistically, CST6 blocks cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation, and single-cell RNA-seq showed CST6 attenuates polarization of monocytes to osteoclast precursors.","method":"ELISA, recombinant protein inhibition assay (cathepsin K activity), ex vivo and in vivo myeloma bone models, single-cell RNA-seq","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mechanistic pathway established with recombinant protein, in vitro enzymatic assay, in vivo models, and single-cell transcriptomics; published in high-tier journal","pmids":["35881476"],"is_preprint":false},{"year":2012,"finding":"CST6 is a secreted cysteine protease inhibitor that suppresses breast cancer cell proliferation, colony formation, migration, invasion, and osteolytic bone metastasis in vivo; the suppressive function against cancer cell motility is carried out by cancer cell-derived soluble CST6.","method":"Label-free secretome proteomics, ectopic expression and knockdown in cancer cells, colony formation assay, migration/invasion assay, animal bone metastasis model","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function and gain-of-function with defined cellular phenotypes, in vivo validation, proteomic identification","pmids":["22688893"],"is_preprint":false},{"year":2014,"finding":"TBX2 transcriptionally represses CST6 via a mechanism co-involving EGR1; CST6 induces apoptosis in TBX2-expressing breast cancer cells by inhibiting legumain (LGMN) intracellularly, not cathepsins — mutation of the LGMN-inhibitory domain of CST6 completely abrogated apoptosis whereas mutation of the cathepsin-inhibitory domain had no effect; CST6 does not require secretion or glycosylation for this intracellular apoptotic effect.","method":"Exogenous CST6 expression in breast cancer cells, domain mutation analysis, LGMN activity assay, siRNA knockdown of LGMN and GPI8, pan-cathepsin inhibitor treatment","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods including domain mutagenesis, enzyme activity assays, and siRNA knockdown in a single study establishing intracellular LGMN inhibition as the mechanism","pmids":["24742492"],"is_preprint":false},{"year":2008,"finding":"Ectopic expression of cystatin E/M (CST6) in glioma cell lines reduced cell motility and invasion, and CST6 is epigenetically silenced by promoter hypermethylation in ~78% of primary brain tumors; CST6 is normally expressed in oligodendrocytes and astrocytes but not neurons.","method":"Ectopic expression in glioma lines, cell motility/invasion assay, methylation-specific PCR, bisulfite genomic sequencing, pyrosequencing, tissue microarray immunohistochemistry","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional loss-of-function and gain-of-function with defined invasion phenotype, epigenetic mechanism confirmed by multiple methylation assays, single lab","pmids":["18607344"],"is_preprint":false},{"year":2006,"finding":"CST6 expression in breast cancer cell lines is silenced by DNA methylation-dependent epigenetic mechanisms; 5-aza-2'-deoxycytidine treatment restores CST6 expression, and bisulfite sequencing confirmed CpG island hypermethylation specifically in the proximal promoter region correlates with gene silencing.","method":"5-aza-2'-deoxycytidine treatment, bisulfite sequencing, methylation-specific PCR, RT-PCR expression analysis in panel of breast cancer cell lines","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — demethylation rescue experiment plus bisulfite sequencing in multiple cell lines, single lab, two orthogonal methods","pmids":["17043665"],"is_preprint":false},{"year":2022,"finding":"A homozygous loss-of-function mutation (p.Gly84Asp) in CST6 causes dry skin, desquamation, and abnormal keratosis; fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on cathepsins (cathepsin V, cathepsin L), and the corresponding mouse mutation resulted in excessive cornification, desquamation, impaired skin barrier, and abnormal keratinocyte proliferation/differentiation.","method":"Whole-exome sequencing, fluorimetric enzyme inhibition assay, mouse knock-in model, histopathology","journal":"Clinical genetics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct enzymatic assay of mutant protein loss-of-function plus in vivo mouse model with defined epidermal phenotype, single lab but multiple orthogonal methods","pmids":["36371786"],"is_preprint":false},{"year":2024,"finding":"Ketamine alleviates breast cancer cell-induced osteoclastogenesis by targeting SRC kinase, which suppresses EGR1 transcription by binding the EGR1 promoter; EGR1 in turn activates CST6 transcription, establishing a SRC/EGR1/CST6 axis where restoration of EGR1 or CST6 counteracts SRC-driven pro-osteoclastic effects.","method":"Co-culture osteoclastogenesis assay, lentiviral overexpression/knockdown, immunoprecipitation (SRC-EGR1 interaction), luciferase reporter assay (EGR1 promoter, CST6 promoter), intracardiac injection mouse model","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunoprecipitation and luciferase assay establish direct transcriptional regulation, in vivo validation included, single lab","pmids":["39695463"],"is_preprint":false},{"year":2024,"finding":"BCMA-CST6 CAR-T cells that secrete CST6 protein suppress osteoclast differentiation and formation (TRAP+ osteoclasts) in vitro and prevent MM-induced bone damage in xenografted mice, while BCMA-CAR-T cells without CST6 do not prevent bone damage despite similar tumor suppression.","method":"CAR-T cell engineering, in vitro osteoclastogenesis assay, xenograft MM mouse model, bioluminescence imaging, micro-CT","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional in vitro and in vivo validation of CST6's bone-protective mechanism via CAR-T delivery, single lab","pmids":["37883186"],"is_preprint":false},{"year":2025,"finding":"In syncytiotrophoblast cells exposed to hypoxia (1% O2), CST6 expression is significantly increased; administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNFα, suggesting CST6 modulates LGMN activity in a context-dependent manner in preeclampsia pathophysiology.","method":"Human trophoblast stem cell differentiation assay, hypoxia exposure (1% O2), recombinant CST6 treatment of endothelial cells, mRNA expression analysis","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited mechanistic follow-up, primarily expression-based with partial functional assay using recombinant protein","pmids":["40234537"],"is_preprint":false}],"current_model":"CST6 (cystatin E/M) is a secreted cysteine protease inhibitor that suppresses cancer cell invasion and bone metastasis by inhibiting cathepsins (particularly CTSB and cathepsin K) and legumain (LGMN) through distinct mechanisms: extracellularly, it enters osteoclasts via endocytosis to suppress CTSB, upregulate SPHK1, and block RANKL-induced p38/NF-κB signaling, thereby inhibiting osteoclastogenesis; intracellularly, it suppresses LGMN to induce apoptosis in cancer cells; its transcription is regulated by a TBX2/EGR1 axis and is frequently silenced by CpG island promoter hypermethylation in breast and brain tumors; loss-of-function mutations ablating cathepsin inhibitory activity cause skin cornification disorders in humans and mice."},"narrative":{"mechanistic_narrative":"CST6 (cystatin E/M) is a secreted cysteine protease inhibitor that restrains tumor cell invasion and osteolytic bone destruction by inhibiting lysosomal cysteine proteases and the asparaginyl endopeptidase legumain through spatially distinct mechanisms [PMID:22688893, PMID:34815788, PMID:24742492]. As a cancer cell-derived soluble factor, it suppresses breast cancer proliferation, migration, invasion and bone metastasis [PMID:22688893]. In the bone microenvironment it acts non-cell-autonomously on osteoclasts: it is endocytosed into osteoclasts where it suppresses cathepsin B, leading to upregulation of its substrate SPHK1 and blockade of RANKL-induced p38 activation, thereby inhibiting osteoclast maturation [PMID:34815788]; in multiple myeloma it inhibits cathepsin K and blocks cathepsin-mediated cleavage of NF-κB/p100 and TRAF3, attenuating monocyte polarization to osteoclast precursors [PMID:35881476]. Intracellularly and independent of secretion or glycosylation, CST6 induces apoptosis in TBX2-expressing breast cancer cells specifically through legumain inhibition rather than cathepsin inhibition [PMID:24742492]. CST6 transcription is controlled by opposing regulators — repressed by TBX2 and activated by EGR1, the latter downstream of an SRC/EGR1 axis — and is frequently silenced by CpG island promoter hypermethylation in breast and brain tumors [PMID:24742492, PMID:39695463, PMID:17043665, PMID:18607344]. A homozygous loss-of-function mutation (p.Gly84Asp) that ablates cathepsin inhibitory activity causes a human skin cornification disorder, recapitulated as excessive cornification and impaired skin barrier in mice [PMID:36371786].","teleology":[{"year":2006,"claim":"Established why CST6 is lost in cancer by showing its silencing is epigenetic rather than mutational, defining a reversible mechanism of tumor suppressor inactivation.","evidence":"5-aza-2'-deoxycytidine rescue and bisulfite sequencing across breast cancer cell lines","pmids":["17043665"],"confidence":"Medium","gaps":["Did not test functional consequences of restored expression","Single lab, cell-line based"]},{"year":2008,"claim":"Generalized the epigenetic silencing and anti-invasive role of CST6 beyond breast tissue to brain tumors, linking promoter hypermethylation to functional loss of motility suppression.","evidence":"Ectopic expression motility/invasion assays plus methylation-specific PCR, bisulfite sequencing, and tissue microarray IHC in gliomas","pmids":["18607344"],"confidence":"Medium","gaps":["Molecular target of motility suppression not identified","No in vivo validation"]},{"year":2012,"claim":"Demonstrated that CST6 is a secreted factor whose soluble form, derived from cancer cells, suppresses invasion and osteolytic bone metastasis, establishing it as a functional metastasis suppressor.","evidence":"Secretome proteomics with gain/loss-of-function and an in vivo bone metastasis model","pmids":["22688893"],"confidence":"High","gaps":["Protease target mediating the anti-metastatic effect not pinpointed","Mechanism of bone protection not dissected"]},{"year":2014,"claim":"Resolved that CST6 has a secretion-independent intracellular function, showing apoptosis induction depends on legumain inhibition rather than cathepsin inhibition, and placed CST6 under TBX2/EGR1 transcriptional control.","evidence":"Domain mutagenesis, LGMN activity assays, and siRNA knockdown in TBX2-expressing breast cancer cells","pmids":["24742492"],"confidence":"High","gaps":["How CST6 accesses intracellular legumain not defined","Apoptotic effectors downstream of LGMN inhibition unresolved"]},{"year":2021,"claim":"Defined a mechanistic pathway for bone protection by showing CST6 is endocytosed into osteoclasts to suppress CTSB, raising SPHK1 and blocking RANKL-induced p38, explaining how it inhibits osteoclastogenesis.","evidence":"Osteoclastogenesis assays, endocytosis tracking, recombinant protein and peptide screening, and preclinical metastasis models","pmids":["34815788"],"confidence":"High","gaps":["Receptor/route of osteoclast endocytosis not identified","Quantitative contribution of SPHK1 axis versus other targets unclear"]},{"year":2022,"claim":"Extended CST6's anti-osteoclastic mechanism to multiple myeloma by showing inhibition of cathepsin K and protection of NF-κB/p100 and TRAF3 from cathepsin cleavage, and linked it to suppressed monocyte-to-osteoclast polarization.","evidence":"Recombinant cathepsin K inhibition assays, ex vivo/in vivo myeloma bone models, and single-cell RNA-seq","pmids":["35881476"],"confidence":"High","gaps":["Which cathepsin directly cleaves p100/TRAF3 in cells not isolated","Relative roles of CTSB versus cathepsin K across contexts unresolved"]},{"year":2022,"claim":"Provided causal human and mouse genetic evidence that CST6 cathepsin-inhibitory activity is required for normal skin cornification, defining a Mendelian disease role.","evidence":"Whole-exome sequencing, fluorimetric inhibition assays of mutant protein against cathepsins V/L, and a mouse knock-in model with histopathology","pmids":["36371786"],"confidence":"High","gaps":["Specific epidermal cathepsin substrate dysregulated in vivo not defined","Single family / single allele"]},{"year":2024,"claim":"Mapped an upstream transcriptional circuit, showing SRC represses EGR1 which activates CST6, providing a druggable node (e.g. ketamine targeting SRC) to restore CST6-mediated bone protection.","evidence":"Co-culture osteoclastogenesis, immunoprecipitation, luciferase reporters of EGR1 and CST6 promoters, and intracardiac injection mouse model","pmids":["39695463"],"confidence":"Medium","gaps":["Direct EGR1 binding to the CST6 promoter not footprinted at base resolution","Generality of the SRC/EGR1/CST6 axis beyond this model untested"]},{"year":2024,"claim":"Demonstrated therapeutic sufficiency of CST6 for bone protection by showing CST6-secreting CAR-T cells prevent myeloma bone damage whereas equivalent CAR-T without CST6 do not, decoupling bone protection from tumor killing.","evidence":"BCMA-CST6 CAR-T engineering, in vitro osteoclastogenesis, and xenograft MM mouse model with micro-CT","pmids":["37883186"],"confidence":"Medium","gaps":["Durability and systemic effects of secreted CST6 not characterized","Single lab proof-of-concept"]},{"year":2025,"claim":"Raised a context-dependent, potentially pro-pathogenic role for CST6, where hypoxia-induced CST6 enhances endothelial dysfunction markers and LGMN expression with TNFα in a preeclampsia model.","evidence":"Trophoblast stem cell differentiation, hypoxia exposure, and recombinant CST6 treatment of endothelial cells with mRNA analysis","pmids":["40234537"],"confidence":"Low","gaps":["Primarily expression-based with limited mechanistic follow-up","Direction of LGMN modulation appears opposite to cancer findings and is unreconciled","No in vivo validation"]},{"year":null,"claim":"How CST6 partitions between its extracellular cathepsin-directed and intracellular legumain-directed functions, and what determines its protective versus pathogenic effect across tissues, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model explaining differential cathepsin versus legumain targeting in vivo","Mechanism of cellular entry/trafficking not defined","Context determinants of pro- versus anti-pathogenic activity unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,3,6]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[0,1,3,6]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2,0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3]}],"complexes":[],"partners":["CTSB","CTSK","LGMN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P04080","full_name":"Cystatin-B","aliases":["CPI-B","Liver thiol proteinase inhibitor","Stefin-B"],"length_aa":98,"mass_kda":11.1,"function":"This is an intracellular thiol proteinase inhibitor. Tightly binding reversible inhibitor of cathepsins L, H and B","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P04080/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CST6","classification":"Not Classified","n_dependent_lines":26,"n_total_lines":1208,"dependency_fraction":0.02152317880794702},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CST6","total_profiled":1310},"omim":[{"mim_id":"618535","title":"ECTODERMAL DYSPLASIA 15, HYPOHIDROTIC/HAIR TYPE; ECTD15","url":"https://www.omim.org/entry/618535"},{"mim_id":"601891","title":"CYSTATIN 6; CST6","url":"https://www.omim.org/entry/601891"},{"mim_id":"601145","title":"CYSTATIN B; CSTB","url":"https://www.omim.org/entry/601145"},{"mim_id":"242500","title":"ICHTHYOSIS, CONGENITAL, AUTOSOMAL RECESSIVE 4B; ARCI4B","url":"https://www.omim.org/entry/242500"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skin 1","ntpm":1026.8}],"url":"https://www.proteinatlas.org/search/CST6"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P04080","domains":[{"cath_id":"3.10.450.10","chopping":"9-89","consensus_level":"high","plddt":97.352,"start":9,"end":89}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P04080","model_url":"https://alphafold.ebi.ac.uk/files/AF-P04080-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P04080-F1-predicted_aligned_error_v6.png","plddt_mean":95.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CST6","jax_strain_url":"https://www.jax.org/strain/search?query=CST6"},"sequence":{"accession":"P04080","fasta_url":"https://rest.uniprot.org/uniprotkb/P04080.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P04080/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P04080"}},"corpus_meta":[{"pmid":"23006792","id":"PMC_23006792","title":"CST6 promoter methylation in circulating cell-free DNA of breast cancer patients.","date":"2012","source":"Clinical biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23006792","citation_count":66,"is_preprint":false},{"pmid":"34815788","id":"PMC_34815788","title":"CST6 protein and peptides inhibit breast cancer bone metastasis by suppressing CTSB activity and osteoclastogenesis.","date":"2021","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/34815788","citation_count":59,"is_preprint":false},{"pmid":"22688893","id":"PMC_22688893","title":"Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis.","date":"2012","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/22688893","citation_count":54,"is_preprint":false},{"pmid":"18607344","id":"PMC_18607344","title":"Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.","date":"2008","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/18607344","citation_count":49,"is_preprint":false},{"pmid":"17043665","id":"PMC_17043665","title":"DNA methylation-dependent silencing of CST6 in human breast cancer cell lines.","date":"2006","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/17043665","citation_count":41,"is_preprint":false},{"pmid":"24742492","id":"PMC_24742492","title":"TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.","date":"2014","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/24742492","citation_count":39,"is_preprint":false},{"pmid":"17540367","id":"PMC_17540367","title":"Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions.","date":"2007","source":"Experimental and molecular pathology","url":"https://pubmed.ncbi.nlm.nih.gov/17540367","citation_count":37,"is_preprint":false},{"pmid":"35881476","id":"PMC_35881476","title":"CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation.","date":"2022","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/35881476","citation_count":23,"is_preprint":false},{"pmid":"12839564","id":"PMC_12839564","title":"The human cystatin M/E gene (CST6): exclusion candidate gene for harlequin ichthyosis.","date":"2003","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/12839564","citation_count":17,"is_preprint":false},{"pmid":"23088560","id":"PMC_23088560","title":"A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.","date":"2012","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/23088560","citation_count":14,"is_preprint":false},{"pmid":"37883186","id":"PMC_37883186","title":"BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions.","date":"2024","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/37883186","citation_count":6,"is_preprint":false},{"pmid":"36371786","id":"PMC_36371786","title":"Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis.","date":"2022","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36371786","citation_count":5,"is_preprint":false},{"pmid":"40234537","id":"PMC_40234537","title":"Cystatin 6 (CST6) and Legumain (LGMN) are potential mediators in the pathogenesis of preeclampsia.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40234537","citation_count":4,"is_preprint":false},{"pmid":"39695463","id":"PMC_39695463","title":"Alleviating role of ketamine in breast cancer cell-induced osteoclastogenesis and tumor bone metastasis-induced bone cancer pain through an SRC/EGR1/CST6 axis.","date":"2024","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39695463","citation_count":4,"is_preprint":false},{"pmid":"38196563","id":"PMC_38196563","title":"Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery.","date":"2024","source":"International journal of general medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38196563","citation_count":3,"is_preprint":false},{"pmid":"39714155","id":"PMC_39714155","title":"Genetic interaction analysis of Candida glabrata transcription factors CST6 and UPC2A in the regulation of respiration and fluconazole susceptibility.","date":"2024","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/39714155","citation_count":2,"is_preprint":false},{"pmid":"42002169","id":"PMC_42002169","title":"Single-cell and spatial transcriptomic profiling reveal CST6 + epithelial-SPP1+ macrophage crosstalk driving lung adenocarcinoma metastasis.","date":"2026","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/42002169","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.26.690797","title":"Role of the  <i>osaA</i>  transcription factor gene in development, secondary metabolism and virulence in the mycotoxigenic fungus  <i>Aspergil lus flavus</i>","date":"2025-11-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.26.690797","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.09.30.679431","title":"Single-cell sequencing of trophoblasts in preeclampsia and chemical hypoxia in BeWo b30 cells reveals EBI3, COL17A1, miR-27a-5p and miR-193b-5p as hypoxia-response markers","date":"2025-10-01","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.30.679431","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.20.25329991","title":"Evaluating the consequences of childhood adiposity on the human plasma proteome at three timepoints across the lifecourse","date":"2025-06-23","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.20.25329991","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13173,"output_tokens":2817,"usd":0.040887,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10137,"output_tokens":3544,"usd":0.069642,"stage2_stop_reason":"end_turn"},"total_usd":0.110529,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"CST6 (cystatin E/M) inhibits osteolytic bone metastasis by entering osteoclasts via endocytosis and suppressing the cysteine protease CTSB (cathepsin B), leading to upregulation of the CTSB hydrolytic substrate SPHK1, which in turn suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, establishing a CST6-CTSB-SPHK1 signaling axis in osteoclast differentiation.\",\n      \"method\": \"In vitro osteoclastogenesis assay, in vivo metastasis assay, endocytosis tracking, recombinant protein treatment, peptide functional screening, preclinical metastasis models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal in vitro and in vivo methods, mechanistic pathway dissected with recombinant protein and functional assays, replicated in preclinical models\",\n      \"pmids\": [\"34815788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CST6 suppresses osteolytic bone disease in multiple myeloma by inhibiting cathepsin K activity and blocking osteoclast differentiation; mechanistically, CST6 blocks cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation, and single-cell RNA-seq showed CST6 attenuates polarization of monocytes to osteoclast precursors.\",\n      \"method\": \"ELISA, recombinant protein inhibition assay (cathepsin K activity), ex vivo and in vivo myeloma bone models, single-cell RNA-seq\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mechanistic pathway established with recombinant protein, in vitro enzymatic assay, in vivo models, and single-cell transcriptomics; published in high-tier journal\",\n      \"pmids\": [\"35881476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CST6 is a secreted cysteine protease inhibitor that suppresses breast cancer cell proliferation, colony formation, migration, invasion, and osteolytic bone metastasis in vivo; the suppressive function against cancer cell motility is carried out by cancer cell-derived soluble CST6.\",\n      \"method\": \"Label-free secretome proteomics, ectopic expression and knockdown in cancer cells, colony formation assay, migration/invasion assay, animal bone metastasis model\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function and gain-of-function with defined cellular phenotypes, in vivo validation, proteomic identification\",\n      \"pmids\": [\"22688893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TBX2 transcriptionally represses CST6 via a mechanism co-involving EGR1; CST6 induces apoptosis in TBX2-expressing breast cancer cells by inhibiting legumain (LGMN) intracellularly, not cathepsins — mutation of the LGMN-inhibitory domain of CST6 completely abrogated apoptosis whereas mutation of the cathepsin-inhibitory domain had no effect; CST6 does not require secretion or glycosylation for this intracellular apoptotic effect.\",\n      \"method\": \"Exogenous CST6 expression in breast cancer cells, domain mutation analysis, LGMN activity assay, siRNA knockdown of LGMN and GPI8, pan-cathepsin inhibitor treatment\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods including domain mutagenesis, enzyme activity assays, and siRNA knockdown in a single study establishing intracellular LGMN inhibition as the mechanism\",\n      \"pmids\": [\"24742492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ectopic expression of cystatin E/M (CST6) in glioma cell lines reduced cell motility and invasion, and CST6 is epigenetically silenced by promoter hypermethylation in ~78% of primary brain tumors; CST6 is normally expressed in oligodendrocytes and astrocytes but not neurons.\",\n      \"method\": \"Ectopic expression in glioma lines, cell motility/invasion assay, methylation-specific PCR, bisulfite genomic sequencing, pyrosequencing, tissue microarray immunohistochemistry\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional loss-of-function and gain-of-function with defined invasion phenotype, epigenetic mechanism confirmed by multiple methylation assays, single lab\",\n      \"pmids\": [\"18607344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CST6 expression in breast cancer cell lines is silenced by DNA methylation-dependent epigenetic mechanisms; 5-aza-2'-deoxycytidine treatment restores CST6 expression, and bisulfite sequencing confirmed CpG island hypermethylation specifically in the proximal promoter region correlates with gene silencing.\",\n      \"method\": \"5-aza-2'-deoxycytidine treatment, bisulfite sequencing, methylation-specific PCR, RT-PCR expression analysis in panel of breast cancer cell lines\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — demethylation rescue experiment plus bisulfite sequencing in multiple cell lines, single lab, two orthogonal methods\",\n      \"pmids\": [\"17043665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A homozygous loss-of-function mutation (p.Gly84Asp) in CST6 causes dry skin, desquamation, and abnormal keratosis; fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on cathepsins (cathepsin V, cathepsin L), and the corresponding mouse mutation resulted in excessive cornification, desquamation, impaired skin barrier, and abnormal keratinocyte proliferation/differentiation.\",\n      \"method\": \"Whole-exome sequencing, fluorimetric enzyme inhibition assay, mouse knock-in model, histopathology\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct enzymatic assay of mutant protein loss-of-function plus in vivo mouse model with defined epidermal phenotype, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"36371786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ketamine alleviates breast cancer cell-induced osteoclastogenesis by targeting SRC kinase, which suppresses EGR1 transcription by binding the EGR1 promoter; EGR1 in turn activates CST6 transcription, establishing a SRC/EGR1/CST6 axis where restoration of EGR1 or CST6 counteracts SRC-driven pro-osteoclastic effects.\",\n      \"method\": \"Co-culture osteoclastogenesis assay, lentiviral overexpression/knockdown, immunoprecipitation (SRC-EGR1 interaction), luciferase reporter assay (EGR1 promoter, CST6 promoter), intracardiac injection mouse model\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunoprecipitation and luciferase assay establish direct transcriptional regulation, in vivo validation included, single lab\",\n      \"pmids\": [\"39695463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BCMA-CST6 CAR-T cells that secrete CST6 protein suppress osteoclast differentiation and formation (TRAP+ osteoclasts) in vitro and prevent MM-induced bone damage in xenografted mice, while BCMA-CAR-T cells without CST6 do not prevent bone damage despite similar tumor suppression.\",\n      \"method\": \"CAR-T cell engineering, in vitro osteoclastogenesis assay, xenograft MM mouse model, bioluminescence imaging, micro-CT\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional in vitro and in vivo validation of CST6's bone-protective mechanism via CAR-T delivery, single lab\",\n      \"pmids\": [\"37883186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In syncytiotrophoblast cells exposed to hypoxia (1% O2), CST6 expression is significantly increased; administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNFα, suggesting CST6 modulates LGMN activity in a context-dependent manner in preeclampsia pathophysiology.\",\n      \"method\": \"Human trophoblast stem cell differentiation assay, hypoxia exposure (1% O2), recombinant CST6 treatment of endothelial cells, mRNA expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited mechanistic follow-up, primarily expression-based with partial functional assay using recombinant protein\",\n      \"pmids\": [\"40234537\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CST6 (cystatin E/M) is a secreted cysteine protease inhibitor that suppresses cancer cell invasion and bone metastasis by inhibiting cathepsins (particularly CTSB and cathepsin K) and legumain (LGMN) through distinct mechanisms: extracellularly, it enters osteoclasts via endocytosis to suppress CTSB, upregulate SPHK1, and block RANKL-induced p38/NF-κB signaling, thereby inhibiting osteoclastogenesis; intracellularly, it suppresses LGMN to induce apoptosis in cancer cells; its transcription is regulated by a TBX2/EGR1 axis and is frequently silenced by CpG island promoter hypermethylation in breast and brain tumors; loss-of-function mutations ablating cathepsin inhibitory activity cause skin cornification disorders in humans and mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CST6 (cystatin E/M) is a secreted cysteine protease inhibitor that restrains tumor cell invasion and osteolytic bone destruction by inhibiting lysosomal cysteine proteases and the asparaginyl endopeptidase legumain through spatially distinct mechanisms [#2, #0, #3]. As a cancer cell-derived soluble factor, it suppresses breast cancer proliferation, migration, invasion and bone metastasis [#2]. In the bone microenvironment it acts non-cell-autonomously on osteoclasts: it is endocytosed into osteoclasts where it suppresses cathepsin B, leading to upregulation of its substrate SPHK1 and blockade of RANKL-induced p38 activation, thereby inhibiting osteoclast maturation [#0]; in multiple myeloma it inhibits cathepsin K and blocks cathepsin-mediated cleavage of NF-\\u03baB/p100 and TRAF3, attenuating monocyte polarization to osteoclast precursors [#1]. Intracellularly and independent of secretion or glycosylation, CST6 induces apoptosis in TBX2-expressing breast cancer cells specifically through legumain inhibition rather than cathepsin inhibition [#3]. CST6 transcription is controlled by opposing regulators \\u2014 repressed by TBX2 and activated by EGR1, the latter downstream of an SRC/EGR1 axis \\u2014 and is frequently silenced by CpG island promoter hypermethylation in breast and brain tumors [#3, #7, #5, #4]. A homozygous loss-of-function mutation (p.Gly84Asp) that ablates cathepsin inhibitory activity causes a human skin cornification disorder, recapitulated as excessive cornification and impaired skin barrier in mice [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established why CST6 is lost in cancer by showing its silencing is epigenetic rather than mutational, defining a reversible mechanism of tumor suppressor inactivation.\",\n      \"evidence\": \"5-aza-2'-deoxycytidine rescue and bisulfite sequencing across breast cancer cell lines\",\n      \"pmids\": [\"17043665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not test functional consequences of restored expression\", \"Single lab, cell-line based\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Generalized the epigenetic silencing and anti-invasive role of CST6 beyond breast tissue to brain tumors, linking promoter hypermethylation to functional loss of motility suppression.\",\n      \"evidence\": \"Ectopic expression motility/invasion assays plus methylation-specific PCR, bisulfite sequencing, and tissue microarray IHC in gliomas\",\n      \"pmids\": [\"18607344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target of motility suppression not identified\", \"No in vivo validation\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that CST6 is a secreted factor whose soluble form, derived from cancer cells, suppresses invasion and osteolytic bone metastasis, establishing it as a functional metastasis suppressor.\",\n      \"evidence\": \"Secretome proteomics with gain/loss-of-function and an in vivo bone metastasis model\",\n      \"pmids\": [\"22688893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease target mediating the anti-metastatic effect not pinpointed\", \"Mechanism of bone protection not dissected\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved that CST6 has a secretion-independent intracellular function, showing apoptosis induction depends on legumain inhibition rather than cathepsin inhibition, and placed CST6 under TBX2/EGR1 transcriptional control.\",\n      \"evidence\": \"Domain mutagenesis, LGMN activity assays, and siRNA knockdown in TBX2-expressing breast cancer cells\",\n      \"pmids\": [\"24742492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CST6 accesses intracellular legumain not defined\", \"Apoptotic effectors downstream of LGMN inhibition unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a mechanistic pathway for bone protection by showing CST6 is endocytosed into osteoclasts to suppress CTSB, raising SPHK1 and blocking RANKL-induced p38, explaining how it inhibits osteoclastogenesis.\",\n      \"evidence\": \"Osteoclastogenesis assays, endocytosis tracking, recombinant protein and peptide screening, and preclinical metastasis models\",\n      \"pmids\": [\"34815788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor/route of osteoclast endocytosis not identified\", \"Quantitative contribution of SPHK1 axis versus other targets unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended CST6's anti-osteoclastic mechanism to multiple myeloma by showing inhibition of cathepsin K and protection of NF-\\u03baB/p100 and TRAF3 from cathepsin cleavage, and linked it to suppressed monocyte-to-osteoclast polarization.\",\n      \"evidence\": \"Recombinant cathepsin K inhibition assays, ex vivo/in vivo myeloma bone models, and single-cell RNA-seq\",\n      \"pmids\": [\"35881476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which cathepsin directly cleaves p100/TRAF3 in cells not isolated\", \"Relative roles of CTSB versus cathepsin K across contexts unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided causal human and mouse genetic evidence that CST6 cathepsin-inhibitory activity is required for normal skin cornification, defining a Mendelian disease role.\",\n      \"evidence\": \"Whole-exome sequencing, fluorimetric inhibition assays of mutant protein against cathepsins V/L, and a mouse knock-in model with histopathology\",\n      \"pmids\": [\"36371786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific epidermal cathepsin substrate dysregulated in vivo not defined\", \"Single family / single allele\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapped an upstream transcriptional circuit, showing SRC represses EGR1 which activates CST6, providing a druggable node (e.g. ketamine targeting SRC) to restore CST6-mediated bone protection.\",\n      \"evidence\": \"Co-culture osteoclastogenesis, immunoprecipitation, luciferase reporters of EGR1 and CST6 promoters, and intracardiac injection mouse model\",\n      \"pmids\": [\"39695463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct EGR1 binding to the CST6 promoter not footprinted at base resolution\", \"Generality of the SRC/EGR1/CST6 axis beyond this model untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated therapeutic sufficiency of CST6 for bone protection by showing CST6-secreting CAR-T cells prevent myeloma bone damage whereas equivalent CAR-T without CST6 do not, decoupling bone protection from tumor killing.\",\n      \"evidence\": \"BCMA-CST6 CAR-T engineering, in vitro osteoclastogenesis, and xenograft MM mouse model with micro-CT\",\n      \"pmids\": [\"37883186\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Durability and systemic effects of secreted CST6 not characterized\", \"Single lab proof-of-concept\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Raised a context-dependent, potentially pro-pathogenic role for CST6, where hypoxia-induced CST6 enhances endothelial dysfunction markers and LGMN expression with TNF\\u03b1 in a preeclampsia model.\",\n      \"evidence\": \"Trophoblast stem cell differentiation, hypoxia exposure, and recombinant CST6 treatment of endothelial cells with mRNA analysis\",\n      \"pmids\": [\"40234537\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Primarily expression-based with limited mechanistic follow-up\", \"Direction of LGMN modulation appears opposite to cancer findings and is unreconciled\", \"No in vivo validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CST6 partitions between its extracellular cathepsin-directed and intracellular legumain-directed functions, and what determines its protective versus pathogenic effect across tissues, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model explaining differential cathepsin versus legumain targeting in vivo\", \"Mechanism of cellular entry/trafficking not defined\", \"Context determinants of pro- versus anti-pathogenic activity unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [0, 1, 3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2, 0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CTSB\", \"CTSK\", \"LGMN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}