{"gene":"CST6","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":2012,"finding":"Secreted CST6 (cystatin E/M) suppresses breast cancer cell proliferation, colony formation, migration, and invasion, and its soluble form inhibits cancer cell motility. Ectopic expression of CST6 in bone-tropic MDA-MB-231 cells rescued mice from osteolytic metastasis, establishing CST6 as a bona fide secreted suppressor of breast cancer bone metastasis.","method":"Secretome proteomic profiling (label-free), ectopic overexpression and knockdown in cancer cell lines, in vitro proliferation/migration/invasion assays, in vivo mouse bone metastasis model","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function with defined cellular and in vivo phenotypes, multiple orthogonal assays, replicated across models","pmids":["22688893"],"is_preprint":false},{"year":2021,"finding":"Cancer cell-derived CST6 enters osteoclast precursors by endocytosis and inhibits the cysteine protease CTSB (cathepsin B), leading to upregulation of the CTSB hydrolytic substrate SPHK1. SPHK1 in turn suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6–CTSB–SPHK1–p38 signaling axis in osteoclast differentiation.","method":"In vitro osteoclastogenesis assay, endocytosis tracking, CTSB activity assay, SPHK1 overexpression/knockdown, p38 phosphorylation analysis, in vivo bone metastasis mouse model, recombinant CST6 protein treatment","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal mechanistic assays (enzyme activity, signaling pathway, endocytosis, in vivo) in a single study with strong controls","pmids":["34815788"],"is_preprint":false},{"year":2008,"finding":"Ectopic expression of CST6/cystatin E/M in glioma cell lines reduced cell motility and invasion, consistent with its role as an invasion suppressor via inhibition of cathepsin B. CST6 is highly expressed in normal oligodendrocytes and astrocytes but not neurons, and its expression is silenced by CpG island hypermethylation in the majority of primary adult and pediatric gliomas, including in tumor-initiating cells.","method":"Cell motility/invasion assays after ectopic CST6 expression, methylation-specific PCR, bisulfite genomic sequencing, pyrosequencing, tissue microarray immunohistochemistry","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2/3 — ectopic expression with functional motility/invasion phenotype; mechanistic link to cathepsin B inferred from family function","pmids":["18607344"],"is_preprint":false},{"year":2014,"finding":"TBX2 (oncogenic transcription factor) represses CST6 transcription via a co-repression mechanism involving EGR1. CST6-induced apoptosis in TBX2-expressing breast cancer cells requires its legumain (LGMN)-inhibitory domain but not its cathepsin-inhibitory domain, identifying LGMN as the key downstream effector. CST6 does not require secretion or glycosylation for this cell-killing function, indicating an intracellular mode of action. siRNA knockdown confirmed LGMN (not GPI8) maintains breast cancer cell proliferation downstream of CST6 loss.","method":"Luciferase reporter assay for TBX2/EGR1-mediated CST6 repression, CST6 domain-specific mutagenesis (LGMN-inhibitory vs cathepsin-inhibitory domains), LGMN activity assay, siRNA knockdown of LGMN and GPI8, apoptosis assay, pan-cathepsin inhibitor treatment","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1–2 — active-site mutagenesis dissecting inhibitory domains, enzyme activity assay, siRNA epistasis, multiple orthogonal methods in one study","pmids":["24742492"],"is_preprint":false},{"year":2022,"finding":"Recombinant CST6 protein inhibits cathepsin K activity and blocks osteoclast differentiation and function in multiple myeloma bone disease. CST6 attenuates monocyte-to-osteoclast-precursor polarization (shown by single-cell RNA-seq) and blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation.","method":"Recombinant protein ELISA, cathepsin K activity assay, ex vivo and in vivo myeloma osteolysis models, single-cell RNA-seq, NF-κB/p100 and TRAF3 cleavage assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 — enzyme activity assay, mechanistic cleavage assays, single-cell transcriptomics, ex vivo and in vivo validation","pmids":["35881476"],"is_preprint":false},{"year":2022,"finding":"Homozygous loss-of-function mutation (p.Gly84Asp) in CST6 causes loss of inhibitory activity against cathepsins V and L and legumain, resulting in dry skin, desquamation, and abnormal keratosis. In mouse knock-in models, the mutation causes excessive cornification, desquamation, impaired skin barrier function, and abnormal keratinocyte proliferation and differentiation.","method":"Whole-exome sequencing, fluorimetric enzyme inhibition assay with cathepsins V/L and legumain, mouse knock-in model with histological and barrier function analysis","journal":"Clinical genetics","confidence":"High","confidence_rationale":"Tier 1–2 — enzyme activity assay with defined mutation, in vivo mouse model with cellular phenotype","pmids":["36371786"],"is_preprint":false},{"year":2024,"finding":"BCMA-CST6 CAR-T cells release CST6 protein upon lysing myeloma cells, and the secreted CST6 suppresses osteoclast differentiation (TRAP+ osteoclast formation). In xenografted MM mice, BCMA-CST6-CAR-T cells (but not BCMA-CAR-T alone) prevented MM-induced bone damage and decreased osteoclast numbers, confirming the functional requirement for CST6 delivery in suppressing osteolysis.","method":"CAR-T cell engineering and in vitro co-culture osteoclastogenesis assay (TRAP staining), bioluminescence imaging of xenograft MM mice, micro-computed tomography of bone","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — defined functional in vitro and in vivo assays; mechanistic pathway previously established, here confirmed in therapeutic context","pmids":["37883186"],"is_preprint":false},{"year":2024,"finding":"Ketamine suppresses SRC kinase activity in breast cancer cells; SRC normally binds the EGR1 promoter to suppress EGR1 transcription. EGR1 in turn activates CST6 transcription. Overexpression of EGR1 or CST6 counteracts SRC-mediated effects, placing CST6 downstream of a SRC–EGR1 transcriptional axis that regulates breast cancer cell viability and osteoclastogenesis.","method":"Immunoprecipitation (SRC-EGR1 promoter binding), luciferase reporter assay (EGR1-CST6 transcriptional regulation), lentiviral overexpression/knockdown, in vitro osteoclastogenesis co-culture, in vivo intracardiac bone metastasis mouse model","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP/IP and luciferase assay defining transcriptional hierarchy; in vivo validation; single study","pmids":["39695463"],"is_preprint":false},{"year":2003,"finding":"The human CST6 gene encodes cystatin M/E, a cysteine proteinase inhibitor expressed in sweat glands, hair follicles, and the stratum granulosum of the epidermis, where it is a putative transglutaminase substrate. Loss-of-function mutations in the mouse Cst6 gene cause the ichq phenotype with abnormal cornification and desquamation, establishing an essential role for CST6 in the final stages of epidermal differentiation. No CST6 mutations were found in 11 harlequin ichthyosis patients, excluding it as a major cause of that condition.","method":"Complete human CST6 gene sequencing, mutation screening of patient cohort by sequencing coding regions and intron-exon boundaries, immunohistochemistry for cystatin M/E protein in patient skin","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2–3 — mouse genetic model with defined skin phenotype; human gene sequencing; protein localization by IHC","pmids":["12839564"],"is_preprint":false},{"year":2006,"finding":"CST6 expression is silenced in breast cancer cell lines through DNA methylation-dependent epigenetic mechanisms. Treatment with 5-aza-2'-deoxycytidine (a DNA methylation inhibitor) restores CST6 expression in CST6-negative lines. Bisulfite sequencing confirmed that hypermethylation of the CST6 proximal promoter CpG island is strongly associated with gene silencing, establishing promoter methylation as the primary mechanism for CST6 loss in breast cancer.","method":"5-aza-2'-deoxycytidine demethylation treatment, bisulfite sequencing, expression analysis by RT-PCR across a panel of breast cancer cell lines","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological demethylation restores expression (functional link), bisulfite sequencing confirms methylation; single laboratory but multiple cell lines","pmids":["17043665"],"is_preprint":false},{"year":2026,"finding":"In vitro co-culture experiments and secretome analysis demonstrated that interaction between CST6-expressing epithelial cells and SPP1+ macrophages in lung adenocarcinoma triggers secretion of pro-metastatic factors TGF-β and MMP9, significantly enhancing tumor cell invasion and migration. Spatial transcriptomics confirmed physical proximity of these two cell subsets at metastatic sites.","method":"In vitro co-culture, secretome analysis (TGF-β, MMP9 measurement), spatial transcriptomics, multiplex immunohistochemistry, single-cell RNA-seq","journal":"International journal of biological macromolecules","confidence":"Low","confidence_rationale":"Tier 3 — co-culture and secretome analysis in a single study; CST6's specific molecular role vs. cell-type marker role not fully resolved","pmids":["42002169"],"is_preprint":false}],"current_model":"CST6 (cystatin M/E) is a secreted cysteine protease inhibitor that suppresses cathepsin B, cathepsin K, cathepsin V/L, and the asparaginyl endopeptidase legumain; it inhibits osteoclastogenesis via a CST6–CTSB–SPHK1–p38 axis and by blocking cathepsin-mediated NF-κB/p100 and TRAF3 cleavage downstream of RANKL, suppresses cancer cell invasion and motility, is transcriptionally repressed by TBX2/EGR1 and SRC/EGR1 signaling axes in cancer, and is required for normal epidermal differentiation through regulation of cathepsin-mediated cornification; its gene is frequently silenced by CpG island promoter hypermethylation in breast cancer and glioma."},"narrative":{"teleology":[{"year":2003,"claim":"The first genetic evidence established that CST6 is essential for terminal epidermal differentiation, resolving whether it has a physiological role beyond protease inhibition in vitro.","evidence":"Mouse Cst6 loss-of-function (ichq phenotype) with abnormal cornification; human gene sequencing and skin IHC","pmids":["12839564"],"confidence":"Medium","gaps":["Specific cathepsin substrates mediating cornification defects were not identified","No human loss-of-function mutation was reported in this study","Mechanism linking CST6 to transglutaminase cross-linking remained uncharacterized"]},{"year":2006,"claim":"Promoter CpG island hypermethylation was identified as the primary mechanism of CST6 silencing in breast cancer, explaining how tumor cells lose this protease inhibitor.","evidence":"Bisulfite sequencing and 5-aza-2'-deoxycytidine demethylation restoring CST6 expression across breast cancer cell lines","pmids":["17043665"],"confidence":"Medium","gaps":["Upstream signals driving de novo methylation at the CST6 locus were not identified","Functional consequence of CST6 re-expression on tumor phenotype was not tested in this study"]},{"year":2008,"claim":"CST6 silencing by promoter methylation was extended to gliomas, and ectopic CST6 expression was shown to reduce glioma cell motility and invasion, broadening its tumor-suppressive role beyond breast cancer.","evidence":"Methylation-specific PCR, bisulfite sequencing, and motility/invasion assays after ectopic CST6 expression in glioma lines","pmids":["18607344"],"confidence":"Medium","gaps":["Direct demonstration that cathepsin B inhibition mediates the invasion-suppressive effect was not provided","In vivo glioma model validation was lacking"]},{"year":2012,"claim":"CST6 was established as a bona fide secreted suppressor of breast cancer bone metastasis, demonstrating that its extracellular protease-inhibitory activity is sufficient to block metastatic colonization.","evidence":"Secretome proteomics, ectopic overexpression/knockdown in MDA-MB-231 cells, in vitro and in vivo bone metastasis models","pmids":["22688893"],"confidence":"High","gaps":["The specific cathepsin target(s) responsible for the bone metastasis phenotype were not dissected","Whether CST6 acts on tumor cells, osteoclasts, or both was unresolved"]},{"year":2014,"claim":"Domain mutagenesis revealed that CST6's anti-tumor activity in TBX2-expressing breast cancer operates through legumain (LGMN) inhibition rather than cathepsin inhibition, and identified TBX2/EGR1 as transcriptional repressors of CST6.","evidence":"Legumain- vs cathepsin-inhibitory domain mutants, LGMN activity assay, siRNA epistasis, luciferase reporter assays for TBX2/EGR1 repression","pmids":["24742492"],"confidence":"High","gaps":["LGMN substrates whose cleavage drives proliferation were not identified","Intracellular vs secreted CST6 trafficking and localization were not fully mapped","Whether the TBX2/EGR1 axis operates across cancer subtypes was untested"]},{"year":2021,"claim":"A full signaling axis was delineated: endocytosed CST6 inhibits cathepsin B in osteoclast precursors, stabilizing SPHK1 and suppressing RANKL-induced p38 activation, explaining how tumor-derived CST6 blocks osteoclastogenesis.","evidence":"Endocytosis tracking, CTSB activity assay, SPHK1 overexpression/knockdown, p38 phosphorylation, in vivo bone metastasis model, recombinant CST6 treatment","pmids":["34815788"],"confidence":"High","gaps":["How CTSB cleavage of SPHK1 is regulated and whether other cathepsins contribute was not resolved","Whether the CST6–CTSB–SPHK1 axis operates in non-cancer osteoclast biology is unknown"]},{"year":2022,"claim":"CST6 was shown to inhibit cathepsin K and block RANKL-dependent cleavage of NF-κB/p100 and TRAF3, defining a second anti-osteoclastogenic mechanism distinct from the SPHK1–p38 axis.","evidence":"Cathepsin K activity assay, NF-κB/p100 and TRAF3 cleavage assays, single-cell RNA-seq of monocyte polarization, ex vivo and in vivo myeloma osteolysis models","pmids":["35881476"],"confidence":"High","gaps":["Relative contributions of cathepsin B vs K inhibition in osteoclast suppression are not quantified","Whether CST6 affects osteoblast activity or only osteoclasts is unaddressed"]},{"year":2022,"claim":"A human CST6 loss-of-function mutation (p.Gly84Asp) was shown to abolish cathepsin V/L and legumain inhibition and cause a Mendelian skin disorder, providing the first human genetic proof that CST6 is required for normal cornification.","evidence":"Whole-exome sequencing, fluorimetric enzyme inhibition assays, mouse knock-in model with histological and barrier function analysis","pmids":["36371786"],"confidence":"High","gaps":["Full spectrum of human CST6-associated skin disease phenotypes remains to be defined","Whether therapeutic protease inhibitors can rescue the skin phenotype was not tested"]},{"year":2024,"claim":"A SRC–EGR1–CST6 transcriptional axis was mapped, revealing that SRC kinase suppresses EGR1 transcription, which in turn controls CST6 expression, connecting oncogenic kinase signaling to CST6 epigenetic/transcriptional silencing.","evidence":"ChIP/IP for SRC–EGR1 promoter binding, luciferase reporter assay, lentiviral overexpression/knockdown, in vivo bone metastasis model","pmids":["39695463"],"confidence":"Medium","gaps":["Whether SRC-mediated CST6 repression cooperates with promoter methylation is unresolved","Single-study finding requiring independent validation"]},{"year":null,"claim":"Key unresolved questions include the structural basis for CST6's dual cathepsin/legumain inhibition, whether CST6 regulates non-canonical (non-cathepsin) pathways in epithelial differentiation, and the therapeutic potential of recombinant CST6 delivery in bone-destructive diseases.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of CST6 in complex with any target protease is available","In vivo pharmacokinetics and delivery strategies for recombinant CST6 are undeveloped","Whether CST6 loss contributes to cancer types beyond breast, glioma, and myeloma is not systematically assessed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3,4,5]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1,4,6]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,2,3]}],"complexes":[],"partners":["CTSB","CTSK","LGMN","SPHK1","TBX2","EGR1"],"other_free_text":[]},"mechanistic_narrative":"CST6 (cystatin M/E) is a secreted cysteine protease inhibitor that targets cathepsins B, K, V/L, and the asparaginyl endopeptidase legumain, functioning as a key regulator of epidermal differentiation, osteoclast biology, and tumor suppression [PMID:22688893, PMID:36371786, PMID:24742492]. In osteoclast precursors, endocytosed CST6 inhibits cathepsin B to stabilize SPHK1, suppressing RANKL-induced p38 signaling, while it also blocks cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 to prevent osteoclast maturation and function [PMID:34815788, PMID:35881476]. Loss-of-function mutation in CST6 (p.Gly84Asp) abolishes cathepsin V/L and legumain inhibition and causes a Mendelian skin disorder with dry skin, desquamation, and abnormal keratosis, consistent with the ichq mouse phenotype [PMID:36371786, PMID:12839564]. CST6 is frequently silenced in breast cancer and glioma by promoter CpG island hypermethylation, and its transcription is repressed by TBX2/EGR1 and SRC/EGR1 signaling axes in cancer cells [PMID:17043665, PMID:18607344, PMID:24742492, PMID:39695463]."},"prefetch_data":{"uniprot":{"accession":"Q15828","full_name":"Cystatin-M","aliases":["Cystatin-6","Cystatin-E"],"length_aa":149,"mass_kda":16.5,"function":"High affinity inhibitor for cathepsin L, cathepsin L2 (cathepsin V), and legumain (PubMed:30425301). Involved in the regulation of epidermal cornification, and hair follicle morphogenesis and maintenance (PubMed:30425301)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q15828/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 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N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8596935","citation_count":456,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"3488317","id":"PMC_3488317","title":"Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids.","date":"1986","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/3488317","citation_count":434,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21145461","id":"PMC_21145461","title":"Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.","date":"2010","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21145461","citation_count":318,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"6203523","id":"PMC_6203523","title":"The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors.","date":"1984","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/6203523","citation_count":282,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22863883","id":"PMC_22863883","title":"A high-throughput approach for measuring temporal changes in the interactome.","date":"2012","source":"Nature methods","url":"https://pubmed.ncbi.nlm.nih.gov/22863883","citation_count":273,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21900206","id":"PMC_21900206","title":"A directed protein interaction network for investigating intracellular signal transduction.","date":"2011","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/21900206","citation_count":258,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19199708","id":"PMC_19199708","title":"Proteomic analysis of human parotid gland exosomes by multidimensional protein identification technology (MudPIT).","date":"2009","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/19199708","citation_count":237,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9769367","id":"PMC_9769367","title":"Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer (review).","date":"1998","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/9769367","citation_count":232,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25037231","id":"PMC_25037231","title":"Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver.","date":"2014","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25037231","citation_count":203,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32203420","id":"PMC_32203420","title":"Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.","date":"2020","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/32203420","citation_count":194,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28675934","id":"PMC_28675934","title":"Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.","date":"2017","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/28675934","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32807901","id":"PMC_32807901","title":"UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination.","date":"2020","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/32807901","citation_count":168,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"1996959","id":"PMC_1996959","title":"Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase.","date":"1991","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/1996959","citation_count":167,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19913121","id":"PMC_19913121","title":"Gene-centric association signals for lipids and apolipoproteins identified via the HumanCVD BeadChip.","date":"2009","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19913121","citation_count":164,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"31995728","id":"PMC_31995728","title":"AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/31995728","citation_count":152,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9090386","id":"PMC_9090386","title":"Unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, EPM1.","date":"1997","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9090386","citation_count":152,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9054946","id":"PMC_9054946","title":"Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPM1.","date":"1997","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9054946","citation_count":147,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12454,"output_tokens":2688,"usd":0.038841},"stage2":{"model":"claude-opus-4-6","input_tokens":6003,"output_tokens":2436,"usd":0.136373},"total_usd":0.402805,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":24613,"output_tokens":4748,"usd":0.072529},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":6940,"output_tokens":2747,"usd":0.155062}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"CST6 (cystatin E/M) is a secreted cysteine protease inhibitor that suppresses breast cancer bone metastasis; cancer cell-derived soluble CST6 suppresses proliferation, colony formation, migration, and invasion of breast cancer cells, and ectopic expression in MDA-MB-231 cells rescued mice from overt osteolytic metastasis.\",\n      \"method\": \"Label-free secretome proteomics, transcriptomic analysis, in vitro functional assays (proliferation, colony formation, migration, invasion), in vivo bone metastasis animal model with ectopic expression and knockdown\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (secretome MS, in vitro assays, in vivo models) in single study with replicated in vivo validation\",\n      \"pmids\": [\"22688893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cancer cell-derived CST6 enters osteoclasts by endocytosis and suppresses the cysteine protease CTSB (cathepsin B), leading to upregulation of the CTSB hydrolytic substrate SPHK1; SPHK1 then suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6-CTSB-SPHK1 signaling axis in osteoclast differentiation.\",\n      \"method\": \"In vitro osteoclastogenesis assays, in vivo metastasis assays, endocytosis experiments, protease activity assays, recombinant protein treatment, in vivo preclinical models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal mechanistic methods (endocytosis, protease assays, pathway interrogation) with in vivo validation\",\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\": \"Recombinant CST6 protein, ELISA, cathepsin K activity assays, 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 — enzymatic activity assay, in vivo model, single-cell transcriptomics, mechanistic pathway dissection in one study\",\n      \"pmids\": [\"35881476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TBX2 transcriptionally represses CST6 via a co-repression mechanism involving EGR1; CST6 acts as a dual protease inhibitor of cathepsins and legumain (LGMN), and its apoptotic function in TBX2-expressing breast cancer cells depends specifically on its LGMN-inhibitory domain (mutation of this domain abrogated apoptosis), while mutation of the cathepsin-inhibitory domain had no effect; CST6 does not require secretion or glycosylation for this intracellular apoptotic activity.\",\n      \"method\": \"ChIP/transcription factor binding assays, site-directed mutagenesis of CST6 domains, LGMN activity assays, siRNA knockdown of LGMN and GPI8, exogenous expression of CST6 in breast cancer cells, pan-cathepsin inhibitor treatment\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of functional domains, enzymatic activity assays, siRNA epistasis, multiple orthogonal methods in single study\",\n      \"pmids\": [\"24742492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CST6 functions as an invasion suppressor in glioma; ectopic expression of cystatin E/M in glioma cell lines reduced cell motility and invasion. CST6 is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. CST6 promoter hypermethylation correlates with reduced/absent expression in 78% of primary brain tumors.\",\n      \"method\": \"Tissue microarray, methylation-specific PCR, bisulfite genomic sequencing, pyrosequencing, ectopic expression with motility/invasion assays\",\n      \"journal\": \"Laboratory investigation; a journal of technical methods and pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — functional invasion assay with ectopic expression; mechanistic link to cathepsin B inhibition inferred from known biology, not directly demonstrated in this paper\",\n      \"pmids\": [\"18607344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CST6 expression is silenced in breast cancer cell lines through DNA methylation-dependent epigenetic mechanisms; treatment with 5-aza-2'-deoxycytidine restored CST6 expression, and bisulfite sequencing confirmed CpG island hypermethylation in the proximal promoter region specifically in CST6-negative cells.\",\n      \"method\": \"5-aza-2'-deoxycytidine treatment, bisulfite sequencing, methylation-specific PCR, correlation of methylation with expression across 12 breast cancer cell lines\",\n      \"journal\": \"Laboratory investigation; a journal of technical methods and pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological demethylation, bisulfite sequencing, and expression profiling across multiple cell lines with strong concordance\",\n      \"pmids\": [\"17043665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss-of-function mutation p.Gly84Asp in CST6 abolishes the inhibitory function of cystatin M/E on cathepsin protease activity, as demonstrated by fluorimetric enzyme assays; the corresponding mouse mutation resulted in excessive cornification, desquamation, impaired skin barrier function, and abnormal proliferation and differentiation of keratinocytes.\",\n      \"method\": \"Whole-exome sequencing, fluorimetric enzyme assays with mutant protein, mouse model with skin phenotype analysis\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct enzymatic assay of mutant protein combined with in vivo mouse model demonstrating functional consequences\",\n      \"pmids\": [\"36371786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SRC kinase suppresses EGR1 transcription by binding the EGR1 promoter; EGR1 in turn activates CST6 transcription; ketamine suppresses SRC, thereby restoring the EGR1/CST6 axis and reducing breast cancer cell osteoclastogenesis and bone metastasis.\",\n      \"method\": \"Immunoprecipitation, luciferase reporter assays for promoter binding, lentiviral overexpression/knockdown, in vitro co-culture osteoclastogenesis, in vivo intracardiac injection mouse model\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — immunoprecipitation and luciferase assays establish molecular interactions; single lab study\",\n      \"pmids\": [\"39695463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BCMA-CST6 CAR-T cells that lyse MM cells and simultaneously release CST6 protein suppress osteoclast differentiation (TRAP+ osteoclast formation) in vitro and prevent MM-induced bone damage in xenografted mice, validating CST6's osteoclast-inhibitory function as therapeutically deliverable.\",\n      \"method\": \"CAR-T cell engineering, in vitro TRAP staining osteoclastogenesis assay, in vivo xenograft MM mouse model with micro-CT imaging\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean in vitro and in vivo functional validation of CST6 anti-osteoclast mechanism via engineered delivery; single study\",\n      \"pmids\": [\"37883186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CST6-encoded cystatin M/E is expressed in sweat glands, hair follicles, and stratum granulosum of human epidermis and presumably acts as a substrate for transglutaminase; a null mutation in the mouse Cst6 gene causes the ichq phenotype with abnormalities in cornification and desquamation, demonstrating an essential role for cystatin M/E in epidermal differentiation.\",\n      \"method\": \"Immunohistochemistry, sequencing of CST6 coding region in ichthyosis patients, mouse null mutation phenotype analysis\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mouse loss-of-function with defined skin phenotype establishes functional role; transglutaminase substrate claim is inferred\",\n      \"pmids\": [\"12839564\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CST6 (cystatin M/E) is a secreted cysteine protease inhibitor that suppresses cathepsins (B, K, L, V) and legumain (LGMN) activity; it inhibits breast cancer metastasis and osteoclastogenesis through a CST6-CTSB-SPHK1 axis (blocking RANKL-induced p38 activation) and a TBX2-EGR1-CST6-LGMN transcriptional pathway, is essential for epidermal differentiation (mouse Cst6 null causes cornification defects), and is frequently silenced in multiple cancers by CpG promoter hypermethylation.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper before extracting discoveries.\n\n**CLASSIFICATION:**\n\nRetrieved papers:\n- [1] CST6 promoter methylation in breast cancer cfDNA → KEEP (but biomarker/expression study, no mechanism) → KEEP but no mechanistic content\n- [2] CST6 inhibits bone metastasis via CTSB-SPHK1-p38 axis → KEEP ✓ mechanistic\n- [3] CST6 as secreted suppressor of breast cancer bone metastasis → KEEP ✓ mechanistic\n- [4] CST6 epigenetic silencing in gliomas → KEEP ✓ (ectopic expression reduces motility/invasion)\n- [5] DNA methylation silencing of CST6 in breast cancer cell lines → KEEP but mostly epigenetic/expression\n- [6] TBX2 represses CST6/LGMN axis → KEEP ✓ mechanistic\n- [7] Methylation-dependent silencing of CST6 → KEEP but expression study\n- [8] CST6 suppresses osteoclast differentiation in myeloma → KEEP ✓ mechanistic\n- [9] Human CST6 gene sequence/harlequin ichthyosis → KEEP ✓ functional\n- [10] MS-HRMA for CST6 methylation → KEEP but no mechanism\n- [11] BCMA-CST6 CAR-T cells → KEEP ✓ mechanistic\n- [12] Loss-of-function CST6 mutations → KEEP ✓ mechanistic\n- [13] Ketamine/SRC/EGR1/CST6 axis → KEEP ✓ mechanistic\n- [14] CST6 and LGMN in preeclampsia → KEEP (some mechanistic)\n- [15] HOXA-AS3/CST6/CBX4 in colon cancer → KEEP but prognostic/expression only\n- [16] Candida glabrata CST6 transcription factor → EXCLUDE (SYMBOL COLLISION — fungal ATF/CREB transcription factor, incompatible with human cysteine protease inhibitor)\n- [17] CST6+ epithelial-SPP1+ macrophage crosstalk in LUAD → KEEP ✓ mechanistic (co-culture experiments)\n- [18] Aspergillus flavus osaA/Cst6 binding motifs → EXCLUDE (SYMBOL COLLISION — fungal transcription factor binding motif)\n- [19] Single-cell sequencing in preeclampsia, mentions CST6 upregulation → KEEP but expression only\n- [20] Childhood adiposity proteome, mentions CST6 → expression association only, no mechanism\n\nGene2pubmed curated papers:\n- [1-6] Large proteomics/interactome studies → not specifically about CST6 mechanism\n- [7] Turk & Bode 1991, cystatins crystal structure → KEEP ✓ mechanistic (foundational for cystatin family, describes stefin B structure and inhibition mechanism — but note this is about cystatin B/stefin B, not CST6/cystatin M/E)\n- [8] Stefin B crystal structure with papain → about stefin B (cystatin B), not CST6 → EXCLUDE (different gene, cystatin B = CSTB not CST6)\n- [9] Chromosome 21 sequence → not mechanistic for CST6\n- [10] Hsp90/CFTR → not CST6\n- [11] Mutations in cystatin B causing EPM1 → EXCLUDE (this is CSTB/cystatin B, not CST6/cystatin M/E — SYMBOL COLLISION: the paper uses \"CST6\" as the gene symbol for cystatin B in older nomenclature)\n- [12] Human cystatin C role → about cystatin C (CST3), not CST6\n- [13] Six cysteine proteinase inhibitors from urine → about cystatins A, B, C, S — not specifically CST6\n- [14] Panorama of metazoan complexes → not CST6 specific\n- [15] CRL ubiquitin ligase network → not CST6\n- [16] Cysteine proteinases and endogenous inhibitors in cancer → general review, no CST6-specific mechanism\n- [17] ECM signatures of colon cancers → not CST6\n- [18] RhoGEF/RhoGAP → not CST6\n- [19] ECM proteomics → not CST6\n- [20] UFMylation of p53 → not CST6\n- [21] Human cystatin C N-terminal segment → about cystatin C (CST3), not CST6\n- [22] Lipids/apolipoproteins GWAS → not CST6\n- [23] AMPK/lysosomal damage → not CST6\n- [24] Minisatellite expansion EPM1 → about cystatin B (CSTB), old nomenclature used CST6 for cystatin B → EXCLUDE (ALIAS COLLISION)\n- [25] Unstable insertion cystatin B gene EPM1 → about cystatin B (CSTB) → EXCLUDE (ALIAS COLLISION)\n\n**Note on gene2pubmed papers 12 (PMID:8596935), 29 (PMID:9090386), 30 (PMID:9054946):** These use \"CST6\" to refer to the cystatin B gene (CSTB) in an older nomenclature. The current gene symbol CST6 refers to cystatin M/E. These are ALIAS COLLISION — EXCLUDE.\n\nNow extracting mechanistic discoveries from KEPT papers:\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"Secreted CST6 (cystatin E/M) suppresses breast cancer cell proliferation, colony formation, migration, and invasion, and its soluble form inhibits cancer cell motility. Ectopic expression of CST6 in bone-tropic MDA-MB-231 cells rescued mice from osteolytic metastasis, establishing CST6 as a bona fide secreted suppressor of breast cancer bone metastasis.\",\n      \"method\": \"Secretome proteomic profiling (label-free), ectopic overexpression and knockdown in cancer cell lines, in vitro proliferation/migration/invasion assays, in vivo mouse bone metastasis model\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function with defined cellular and in vivo phenotypes, multiple orthogonal assays, replicated across models\",\n      \"pmids\": [\"22688893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cancer cell-derived CST6 enters osteoclast precursors by endocytosis and inhibits the cysteine protease CTSB (cathepsin B), leading to upregulation of the CTSB hydrolytic substrate SPHK1. SPHK1 in turn suppresses osteoclast maturation by inhibiting RANKL-induced p38 activation, defining a CST6–CTSB–SPHK1–p38 signaling axis in osteoclast differentiation.\",\n      \"method\": \"In vitro osteoclastogenesis assay, endocytosis tracking, CTSB activity assay, SPHK1 overexpression/knockdown, p38 phosphorylation analysis, in vivo bone metastasis mouse model, recombinant CST6 protein treatment\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal mechanistic assays (enzyme activity, signaling pathway, endocytosis, in vivo) in a single study with strong controls\",\n      \"pmids\": [\"34815788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ectopic expression of CST6/cystatin E/M in glioma cell lines reduced cell motility and invasion, consistent with its role as an invasion suppressor via inhibition of cathepsin B. CST6 is highly expressed in normal oligodendrocytes and astrocytes but not neurons, and its expression is silenced by CpG island hypermethylation in the majority of primary adult and pediatric gliomas, including in tumor-initiating cells.\",\n      \"method\": \"Cell motility/invasion assays after ectopic CST6 expression, methylation-specific PCR, bisulfite genomic sequencing, pyrosequencing, tissue microarray immunohistochemistry\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — ectopic expression with functional motility/invasion phenotype; mechanistic link to cathepsin B inferred from family function\",\n      \"pmids\": [\"18607344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TBX2 (oncogenic transcription factor) represses CST6 transcription via a co-repression mechanism involving EGR1. CST6-induced apoptosis in TBX2-expressing breast cancer cells requires its legumain (LGMN)-inhibitory domain but not its cathepsin-inhibitory domain, identifying LGMN as the key downstream effector. CST6 does not require secretion or glycosylation for this cell-killing function, indicating an intracellular mode of action. siRNA knockdown confirmed LGMN (not GPI8) maintains breast cancer cell proliferation downstream of CST6 loss.\",\n      \"method\": \"Luciferase reporter assay for TBX2/EGR1-mediated CST6 repression, CST6 domain-specific mutagenesis (LGMN-inhibitory vs cathepsin-inhibitory domains), LGMN activity assay, siRNA knockdown of LGMN and GPI8, apoptosis assay, pan-cathepsin inhibitor treatment\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — active-site mutagenesis dissecting inhibitory domains, enzyme activity assay, siRNA epistasis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"24742492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Recombinant CST6 protein inhibits cathepsin K activity and blocks osteoclast differentiation and function in multiple myeloma bone disease. CST6 attenuates monocyte-to-osteoclast-precursor polarization (shown by single-cell RNA-seq) and blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation.\",\n      \"method\": \"Recombinant protein ELISA, cathepsin K activity assay, ex vivo and in vivo myeloma osteolysis models, single-cell RNA-seq, NF-κB/p100 and TRAF3 cleavage assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — enzyme activity assay, mechanistic cleavage assays, single-cell transcriptomics, ex vivo and in vivo validation\",\n      \"pmids\": [\"35881476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Homozygous loss-of-function mutation (p.Gly84Asp) in CST6 causes loss of inhibitory activity against cathepsins V and L and legumain, resulting in dry skin, desquamation, and abnormal keratosis. In mouse knock-in models, the mutation causes excessive cornification, desquamation, impaired skin barrier function, and abnormal keratinocyte proliferation and differentiation.\",\n      \"method\": \"Whole-exome sequencing, fluorimetric enzyme inhibition assay with cathepsins V/L and legumain, mouse knock-in model with histological and barrier function analysis\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — enzyme activity assay with defined mutation, in vivo mouse model with cellular phenotype\",\n      \"pmids\": [\"36371786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BCMA-CST6 CAR-T cells release CST6 protein upon lysing myeloma cells, and the secreted CST6 suppresses osteoclast differentiation (TRAP+ osteoclast formation). In xenografted MM mice, BCMA-CST6-CAR-T cells (but not BCMA-CAR-T alone) prevented MM-induced bone damage and decreased osteoclast numbers, confirming the functional requirement for CST6 delivery in suppressing osteolysis.\",\n      \"method\": \"CAR-T cell engineering and in vitro co-culture osteoclastogenesis assay (TRAP staining), bioluminescence imaging of xenograft MM mice, micro-computed tomography of bone\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined functional in vitro and in vivo assays; mechanistic pathway previously established, here confirmed in therapeutic context\",\n      \"pmids\": [\"37883186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ketamine suppresses SRC kinase activity in breast cancer cells; SRC normally binds the EGR1 promoter to suppress EGR1 transcription. EGR1 in turn activates CST6 transcription. Overexpression of EGR1 or CST6 counteracts SRC-mediated effects, placing CST6 downstream of a SRC–EGR1 transcriptional axis that regulates breast cancer cell viability and osteoclastogenesis.\",\n      \"method\": \"Immunoprecipitation (SRC-EGR1 promoter binding), luciferase reporter assay (EGR1-CST6 transcriptional regulation), lentiviral overexpression/knockdown, in vitro osteoclastogenesis co-culture, in vivo intracardiac bone metastasis mouse model\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP/IP and luciferase assay defining transcriptional hierarchy; in vivo validation; single study\",\n      \"pmids\": [\"39695463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human CST6 gene encodes cystatin M/E, a cysteine proteinase inhibitor expressed in sweat glands, hair follicles, and the stratum granulosum of the epidermis, where it is a putative transglutaminase substrate. Loss-of-function mutations in the mouse Cst6 gene cause the ichq phenotype with abnormal cornification and desquamation, establishing an essential role for CST6 in the final stages of epidermal differentiation. No CST6 mutations were found in 11 harlequin ichthyosis patients, excluding it as a major cause of that condition.\",\n      \"method\": \"Complete human CST6 gene sequencing, mutation screening of patient cohort by sequencing coding regions and intron-exon boundaries, immunohistochemistry for cystatin M/E protein in patient skin\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — mouse genetic model with defined skin phenotype; human gene sequencing; protein localization by IHC\",\n      \"pmids\": [\"12839564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CST6 expression is silenced in breast cancer cell lines through DNA methylation-dependent epigenetic mechanisms. Treatment with 5-aza-2'-deoxycytidine (a DNA methylation inhibitor) restores CST6 expression in CST6-negative lines. Bisulfite sequencing confirmed that hypermethylation of the CST6 proximal promoter CpG island is strongly associated with gene silencing, establishing promoter methylation as the primary mechanism for CST6 loss in breast cancer.\",\n      \"method\": \"5-aza-2'-deoxycytidine demethylation treatment, bisulfite sequencing, expression analysis by RT-PCR across a panel of breast cancer cell lines\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological demethylation restores expression (functional link), bisulfite sequencing confirms methylation; single laboratory but multiple cell lines\",\n      \"pmids\": [\"17043665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In vitro co-culture experiments and secretome analysis demonstrated that interaction between CST6-expressing epithelial cells and SPP1+ macrophages in lung adenocarcinoma triggers secretion of pro-metastatic factors TGF-β and MMP9, significantly enhancing tumor cell invasion and migration. Spatial transcriptomics confirmed physical proximity of these two cell subsets at metastatic sites.\",\n      \"method\": \"In vitro co-culture, secretome analysis (TGF-β, MMP9 measurement), spatial transcriptomics, multiplex immunohistochemistry, single-cell RNA-seq\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-culture and secretome analysis in a single study; CST6's specific molecular role vs. cell-type marker role not fully resolved\",\n      \"pmids\": [\"42002169\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CST6 (cystatin M/E) is a secreted cysteine protease inhibitor that suppresses cathepsin B, cathepsin K, cathepsin V/L, and the asparaginyl endopeptidase legumain; it inhibits osteoclastogenesis via a CST6–CTSB–SPHK1–p38 axis and by blocking cathepsin-mediated NF-κB/p100 and TRAF3 cleavage downstream of RANKL, suppresses cancer cell invasion and motility, is transcriptionally repressed by TBX2/EGR1 and SRC/EGR1 signaling axes in cancer, and is required for normal epidermal differentiation through regulation of cathepsin-mediated cornification; its gene is frequently silenced by CpG island promoter hypermethylation in breast cancer and glioma.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CST6 encodes cystatin M/E, a secreted cysteine protease inhibitor that functions as a broad suppressor of cathepsins (B, K, L, V) and legumain (LGMN), with essential roles in epidermal homeostasis and tumor suppression. In bone biology, cancer cell-derived CST6 is endocytosed by osteoclasts and suppresses cathepsin B, stabilizing its substrate SPHK1 and thereby inhibiting RANKL-induced p38 activation and osteoclast maturation; CST6 also blocks cathepsin K-mediated cleavage of NF-κB/p100 and TRAF3 to prevent osteoclast differentiation in multiple myeloma [PMID:34815788, PMID:35881476]. CST6's intracellular pro-apoptotic activity in breast cancer cells depends specifically on its legumain-inhibitory domain rather than its cathepsin-inhibitory domain, and its expression is transcriptionally repressed by TBX2 via EGR1 and silenced by CpG promoter hypermethylation across multiple cancer types [PMID:24742492, PMID:17043665]. Loss-of-function mutations in CST6 abolish cathepsin inhibition and cause ichthyosis-like defects in cornification, desquamation, and keratinocyte differentiation, establishing CST6 as essential for epidermal barrier function [PMID:36371786, PMID:12839564].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that CST6 is essential for normal epidermal differentiation resolved why cystatin M/E is expressed in the granular layer and hair follicles, and defined the first organismal consequence of its loss.\",\n      \"evidence\": \"Mouse Cst6 null mutation producing the ichq phenotype with cornification/desquamation defects; immunohistochemistry of human epidermis\",\n      \"pmids\": [\"12839564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transglutaminase substrate role is inferred, not directly demonstrated\", \"Specific cathepsin targets in epidermis not identified in this study\", \"No human disease-causing mutation reported at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that CST6 silencing in breast cancer occurs through promoter CpG hypermethylation established an epigenetic mechanism for loss of this protease inhibitor in tumors.\",\n      \"evidence\": \"Demethylation with 5-aza-2'-deoxycytidine restored CST6 expression; bisulfite sequencing confirmed promoter methylation across 12 breast cancer cell lines\",\n      \"pmids\": [\"17043665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Methylation-expression correlation in primary tumors not extensively shown\", \"Upstream signals driving methylation not identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that ectopic CST6 expression suppresses glioma invasion extended its tumor-suppressive function beyond breast cancer and linked promoter methylation to silencing in brain tumors.\",\n      \"evidence\": \"Ectopic expression reduced glioma cell motility/invasion; methylation-specific PCR and pyrosequencing of primary brain tumors\",\n      \"pmids\": [\"18607344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct cathepsin B inhibition not demonstrated in glioma cells in this study\", \"In vivo glioma model not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that cancer cell-secreted CST6 suppresses breast cancer bone metastasis in vivo established CST6 as a paracrine metastasis suppressor acting on the bone microenvironment.\",\n      \"evidence\": \"Secretome proteomics, ectopic CST6 in MDA-MB-231 cells rescuing mice from osteolytic metastasis, knockdown increasing metastasis\",\n      \"pmids\": [\"22688893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of CST6 action in bone niche not yet defined\", \"Specific protease target in bone cells not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Dissecting CST6's dual inhibitory domains revealed that the legumain-inhibitory domain, not the cathepsin-inhibitory domain, mediates intracellular apoptosis in breast cancer cells, and that TBX2-EGR1 transcriptionally represses CST6.\",\n      \"evidence\": \"Site-directed mutagenesis of CST6 inhibitory domains, LGMN activity assays, ChIP for TBX2/EGR1 binding, siRNA epistasis\",\n      \"pmids\": [\"24742492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How LGMN inhibition triggers apoptosis mechanistically is not resolved\", \"GPI8 involvement suggested but not deeply characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying the CST6→CTSB→SPHK1 signaling axis in osteoclasts explained how paracrine CST6 suppresses osteoclastogenesis: CST6 enters osteoclasts, inhibits cathepsin B, stabilizes SPHK1, and blocks RANKL-induced p38 activation.\",\n      \"evidence\": \"Endocytosis experiments, protease activity assays, recombinant CST6 treatment, in vivo metastasis models\",\n      \"pmids\": [\"34815788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPHK1 stabilization is the sole mechanism or additional cathepsin substrates contribute is unclear\", \"Endocytic receptor for CST6 uptake not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extending CST6's anti-osteoclast mechanism to multiple myeloma revealed that CST6 inhibits cathepsin K and blocks cleavage of NF-κB/p100 and TRAF3, while single-cell RNA-seq showed attenuated monocyte-to-osteoclast polarization.\",\n      \"evidence\": \"Cathepsin K activity assays, ex vivo and in vivo myeloma bone models, scRNA-seq\",\n      \"pmids\": [\"35881476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of cathepsin B versus cathepsin K inhibition in different disease contexts not resolved\", \"Whether CST6 also affects osteoblast function is unexplored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A human CST6 loss-of-function mutation (p.Gly84Asp) that abolishes cathepsin inhibitory activity in vitro and causes ichthyosis-like skin disease in mice provided direct biochemical and genetic proof that CST6 protease inhibition is required for epidermal barrier integrity.\",\n      \"evidence\": \"Whole-exome sequencing, fluorimetric enzyme assays with mutant protein, mouse model skin phenotype\",\n      \"pmids\": [\"36371786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Human patient phenotype not fully characterized in this report\", \"Structural basis for Gly84Asp loss of function not determined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defining the SRC→EGR1→CST6 transcriptional axis and engineering BCMA-CST6 CAR-T cells showed that CST6 expression is regulated by SRC kinase and that therapeutic delivery of CST6 protein is feasible for bone-protective therapy.\",\n      \"evidence\": \"Luciferase reporter assays, lentiviral overexpression/knockdown, CAR-T engineering, in vivo xenograft MM model with micro-CT\",\n      \"pmids\": [\"39695463\", \"37883186\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SRC-EGR1 regulation described in single lab study; independent replication needed\", \"Long-term efficacy and specificity of CST6-releasing CAR-T not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The endocytic receptor mediating CST6 uptake into osteoclasts, the structural basis for selective legumain versus cathepsin inhibition in apoptosis, and the upstream signals driving CST6 promoter hypermethylation in cancer remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Endocytic receptor for CST6 internalization not identified\", \"Structural determinants of dual inhibitory specificity not resolved at atomic level\", \"Epigenetic writers responsible for CST6 promoter methylation in tumorigenesis unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 3, 6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 2, 4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CTSB\", \"CTSK\", \"LGMN\", \"SPHK1\", \"TBX2\", \"EGR1\", \"SRC\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CST6 (cystatin M/E) is a secreted cysteine protease inhibitor that targets cathepsins B, K, V/L, and the asparaginyl endopeptidase legumain, functioning as a key regulator of epidermal differentiation, osteoclast biology, and tumor suppression [PMID:22688893, PMID:36371786, PMID:24742492]. In osteoclast precursors, endocytosed CST6 inhibits cathepsin B to stabilize SPHK1, suppressing RANKL-induced p38 signaling, while it also blocks cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 to prevent osteoclast maturation and function [PMID:34815788, PMID:35881476]. Loss-of-function mutation in CST6 (p.Gly84Asp) abolishes cathepsin V/L and legumain inhibition and causes a Mendelian skin disorder with dry skin, desquamation, and abnormal keratosis, consistent with the ichq mouse phenotype [PMID:36371786, PMID:12839564]. CST6 is frequently silenced in breast cancer and glioma by promoter CpG island hypermethylation, and its transcription is repressed by TBX2/EGR1 and SRC/EGR1 signaling axes in cancer cells [PMID:17043665, PMID:18607344, PMID:24742492, PMID:39695463].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"The first genetic evidence established that CST6 is essential for terminal epidermal differentiation, resolving whether it has a physiological role beyond protease inhibition in vitro.\",\n      \"evidence\": \"Mouse Cst6 loss-of-function (ichq phenotype) with abnormal cornification; human gene sequencing and skin IHC\",\n      \"pmids\": [\"12839564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific cathepsin substrates mediating cornification defects were not identified\",\n        \"No human loss-of-function mutation was reported in this study\",\n        \"Mechanism linking CST6 to transglutaminase cross-linking remained uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Promoter CpG island hypermethylation was identified as the primary mechanism of CST6 silencing in breast cancer, explaining how tumor cells lose this protease inhibitor.\",\n      \"evidence\": \"Bisulfite sequencing and 5-aza-2'-deoxycytidine demethylation restoring CST6 expression across breast cancer cell lines\",\n      \"pmids\": [\"17043665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Upstream signals driving de novo methylation at the CST6 locus were not identified\",\n        \"Functional consequence of CST6 re-expression on tumor phenotype was not tested in this study\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"CST6 silencing by promoter methylation was extended to gliomas, and ectopic CST6 expression was shown to reduce glioma cell motility and invasion, broadening its tumor-suppressive role beyond breast cancer.\",\n      \"evidence\": \"Methylation-specific PCR, bisulfite sequencing, and motility/invasion assays after ectopic CST6 expression in glioma lines\",\n      \"pmids\": [\"18607344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct demonstration that cathepsin B inhibition mediates the invasion-suppressive effect was not provided\",\n        \"In vivo glioma model validation was lacking\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"CST6 was established as a bona fide secreted suppressor of breast cancer bone metastasis, demonstrating that its extracellular protease-inhibitory activity is sufficient to block metastatic colonization.\",\n      \"evidence\": \"Secretome proteomics, ectopic overexpression/knockdown in MDA-MB-231 cells, in vitro and in vivo bone metastasis models\",\n      \"pmids\": [\"22688893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific cathepsin target(s) responsible for the bone metastasis phenotype were not dissected\",\n        \"Whether CST6 acts on tumor cells, osteoclasts, or both was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Domain mutagenesis revealed that CST6's anti-tumor activity in TBX2-expressing breast cancer operates through legumain (LGMN) inhibition rather than cathepsin inhibition, and identified TBX2/EGR1 as transcriptional repressors of CST6.\",\n      \"evidence\": \"Legumain- vs cathepsin-inhibitory domain mutants, LGMN activity assay, siRNA epistasis, luciferase reporter assays for TBX2/EGR1 repression\",\n      \"pmids\": [\"24742492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"LGMN substrates whose cleavage drives proliferation were not identified\",\n        \"Intracellular vs secreted CST6 trafficking and localization were not fully mapped\",\n        \"Whether the TBX2/EGR1 axis operates across cancer subtypes was untested\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A full signaling axis was delineated: endocytosed CST6 inhibits cathepsin B in osteoclast precursors, stabilizing SPHK1 and suppressing RANKL-induced p38 activation, explaining how tumor-derived CST6 blocks osteoclastogenesis.\",\n      \"evidence\": \"Endocytosis tracking, CTSB activity assay, SPHK1 overexpression/knockdown, p38 phosphorylation, in vivo bone metastasis model, recombinant CST6 treatment\",\n      \"pmids\": [\"34815788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How CTSB cleavage of SPHK1 is regulated and whether other cathepsins contribute was not resolved\",\n        \"Whether the CST6–CTSB–SPHK1 axis operates in non-cancer osteoclast biology is unknown\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CST6 was shown to inhibit cathepsin K and block RANKL-dependent cleavage of NF-κB/p100 and TRAF3, defining a second anti-osteoclastogenic mechanism distinct from the SPHK1–p38 axis.\",\n      \"evidence\": \"Cathepsin K activity assay, NF-κB/p100 and TRAF3 cleavage assays, single-cell RNA-seq of monocyte polarization, ex vivo and in vivo myeloma osteolysis models\",\n      \"pmids\": [\"35881476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Relative contributions of cathepsin B vs K inhibition in osteoclast suppression are not quantified\",\n        \"Whether CST6 affects osteoblast activity or only osteoclasts is unaddressed\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A human CST6 loss-of-function mutation (p.Gly84Asp) was shown to abolish cathepsin V/L and legumain inhibition and cause a Mendelian skin disorder, providing the first human genetic proof that CST6 is required for normal cornification.\",\n      \"evidence\": \"Whole-exome sequencing, fluorimetric enzyme inhibition assays, mouse knock-in model with histological and barrier function analysis\",\n      \"pmids\": [\"36371786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full spectrum of human CST6-associated skin disease phenotypes remains to be defined\",\n        \"Whether therapeutic protease inhibitors can rescue the skin phenotype was not tested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A SRC–EGR1–CST6 transcriptional axis was mapped, revealing that SRC kinase suppresses EGR1 transcription, which in turn controls CST6 expression, connecting oncogenic kinase signaling to CST6 epigenetic/transcriptional silencing.\",\n      \"evidence\": \"ChIP/IP for SRC–EGR1 promoter binding, luciferase reporter assay, lentiviral overexpression/knockdown, in vivo bone metastasis model\",\n      \"pmids\": [\"39695463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether SRC-mediated CST6 repression cooperates with promoter methylation is unresolved\",\n        \"Single-study finding requiring independent validation\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for CST6's dual cathepsin/legumain inhibition, whether CST6 regulates non-canonical (non-cathepsin) pathways in epithelial differentiation, and the therapeutic potential of recombinant CST6 delivery in bone-destructive diseases.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No crystal structure of CST6 in complex with any target protease is available\",\n        \"In vivo pharmacokinetics and delivery strategies for recombinant CST6 are undeveloped\",\n        \"Whether CST6 loss contributes to cancer types beyond breast, glioma, and myeloma is not systematically assessed\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 4, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CTSB\",\n      \"CTSK\",\n      \"LGMN\",\n      \"SPHK1\",\n      \"TBX2\",\n      \"EGR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}