{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/checksum.txt"],"Mzml":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250606_MAvila_2348_TMTpro_10Fr_Static.mzML"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_9_2348_TMTpro_MS2v3_Fr_3.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_3_2348_TMTpro_MS2v3_Fr_1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_24_2348_TMTpro_MS2v3_Fr_8.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_6_2348_TMTpro_MS2v3_Fr_2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_27_2348_TMTpro_MS2v3_Fr_9.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_30_2348_TMTpro_MS2v3_Fr_10.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_12_2348_TMTpro_MS2v3_Fr_4.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_18_2348_TMTpro_MS2v3_Fr_6.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_21_2348_TMTpro_MS2v3_Fr_7.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250604_15_2348_TMTpro_MS2v3_Fr_5.raw"],"Mzid":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD072211/20250606_MAvila_2348_TMTpro_10Fr_Static.mzid"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["maavila@unav.es"],"submitter":["Sergio Ciordia"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"disease":["Cholangiocarcinoma"],"software":[""],"submitter_keywords":["Drug targets","Mouse models","Epigenetics","Metabolism","Cholangiocarcinoma"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD072211"],"tissue":["Liver"],"sample_protocol":["Liver tissues were mechanically disrupted using a pellet pestle cordless motor in 200 μl ice-cold RIPA buffer (30 mM Tris pH 7.5, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 5mM EDTA, 1% NP-40, 1% Triton-X 100, 3.6% B-glycerophosphate, 0.5% sodium deoxycholate, 10 mM sodium fluoride and 1mM sodium orthovanadate, all purchased from Sigma) supplemented 1x protease inhibitors (Complete Mini Protease Inhibitor Cocktail, Roche). The lysates were subsequently clarified by ultracentrifugation at 100,000 ×g for 30 minutes at 4°C using a Hitachi ultracentrifuge. The resulting supernatant, corresponding to the soluble protein fraction, was carefully collected and transferred to fresh tubes for the subsequent precipitation in Methanol/Chloroform. The pellets were resuspended in 200 µL of 2.5% (w/v) SDS, 25 mM Triethylammonium bicarbonate (TEAB), 5 mM TCEP, and 10 mM chloroacetamide (CAA) supplemented with Pierce™ DNase (25 kU, 88701, ThermoFisher). Lysates were incubated at 60°C for 30 min to reduce and alkylate protein cysteine residues, followed by sonication using an ultrasonic processor UP50H (Hielscher Ultrasonics) for 1 min on ice (0.5 cycles, 100% amplitude). The protein extracts were centrifuged at 18,400 ×g for 10 min, and the supernatant was transferred to a new tube and quantified using Pierce™ 660 nm Protein Assay Reagent supplemented with Ionic Detergent Compatibility Reagent according to the manufacturer’s instructions. Automated SP3-based protein digestion was performed on the Opentrons OT-2 platform in the presence of MagReSyn® Hydroxy microparticles as described elsewhere (82), with the corresponding adaptations for our sample format. Proteins were digested overnight at 37°C using trypsin (1:33, enz:prot) and Lys-C (1:500, enz:prot). Eluted peptides were dried by speed vacuum and quantified by fluorimetry (Qubit). Peptide samples were labeled using the TMT-18plex Isobaric Mass Tagging Kit according to the manufacturer’s instructions, including two pooled internal standards labeled with the 133C and 133N channels. After labeling, samples were pooled, evaporated to dryness, and stored at -20°C. High-pH reversed-phase fractionation was performed using Styrene Divinylbenzene reverse phase sulfonate (SDB-RPS) StageTips (CDS Empore™, Sigma-Aldrich) as previously reported (82), applying stepwise elution with increasing acetonitrile (ACN) concentrations (0-60%) in 10 mM ammonium formate (pH 10.0) to obtain five fractions, which were dried and stored at -20 °C. For nano-Liquid Chromatography coupled to Electrospray Ionization Tandem Mass Spectrometry (nanoLC-ESI-MS/MS) analysis, 500 ng of each high-pH fraction was injected into an Ultimate 3000 nano-HPLC system (Thermo Fisher Scientific) connected online to an Orbitrap Exploris™ 240 mass spectrometer as previously reported (82). Briefly, peptides were separated on an Easy-spray PepMap C18 analytical column. MS1 spectra were acquired at 60,000 resolution (m/z 200), followed by data-dependent selection of the top 20 precursors for higher-energy collisional dissociation fragmentation (34% NCE). MS2 spectra were acquired at 45,000 resolution with an automatic gain control (AGC) target of 50%, using a 0.7 m/z isolation window and a 45-s dynamic exclusion. Precursor ions with charge states from +2 to +5 were selected for fragmentation."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw data files were processed using the Proteome Discoverer 2.5.0.400 software (Thermo Scientific, Bremen, Germany), and a database search was carried out using using three search engines (Mascot (v2.8.0), MsFragger (v3.1.1), and Sequest HT) against Mus musculus UniProtKB reviewed database (14th September 2024, 20,470 sequences) containing the most common laboratory contaminants (cRAP database with 70 sequences). Search parameters were set as follows: cysteine carbamidomethyl (+57.021464 Da) and TMTpro (+304.207 Da) on lysine and N-term as fixed modifications; methionine oxidation (+15.995 Da), N-term acetylation (+42.010 Da), and Gln→pyro-Glu (-17.026 Da) as variable modifications. Precursor mass tolerances were set at 10 ppm and the fragment mass tolerance at 0.02 Da and trypsin/P was selected as a protease with a maximum of 2 missed cleavage sites. False discovery rate (FDR) was calculated using the processing node Percolator (maximum delta Cn 0.05; decoy database search target) and the validation of proteins, peptides, and peptide spectral matches (PSMs) peptides with an FDR≤1 %. The quantitation was also performed in Proteome Discoverer using the “Reporter Ions Quantifier” feature in the quantification workflow using the following parameters: unique+razor peptides were used for quantitation, co-isolation threshold was set at 50%, signal to noise of reporter ions was 10, and the normalization and scaling were performed considering the total peptide amount and the control (IS) average, respectively. The protein ratio was calculated considering the protein abundance and the hypothesis test was based on a “t-test (background-based)”. Protein groups (master proteins) with an FDR lower than 1% and with abundance values in both IS were considered for quantitation. To identify the proteins that were differentially expressed in each comparison, an adjusted p-value threshold of ≤ 0.05 was applied using the Benjamini-Hochberg post hoc adjustment. Volcano plot and Principal Component Analysis (PCA) were performed in Proteome Discover considering the differentially expressed proteins in each comparison."],"omics_type":["Proteomics"],"labhead":["Matias A Avila"],"instrument_platform":[""],"labhead_affiliation":["Hepatology Laboratory, Solid Tumors Program, CIMA, CCUN, University of Navarra (Pamplona). Spain"],"submission_type":["COMPLETE"],"species":["Mus Musculus (mouse)"],"submitter_mail":["sciordia@cnb.csic.es"],"publication":["42326014 Lopez-Pascual A, Elurbide J, Valbuena-Goiricelaya E, Latasa MU, Anaya E, Adan-Villaescusa E, Castelló-Uribe B, Martínez-Pérez LA, Uriarte I, Arechederra M, Ciordia S, Corrales FJ, Strnad P, Frankova S, Sticova E, Fabian O, Colyn L, Inacio P, Bayo J, Huch M, Berasain C, Fernández-Barrena MG, Avila MA. Transcriptomic profiling of epigenetic regulators and metabolic reprogramming in human cholangiocarcinoma. Front Cell Dev Biol. 2026 14:1765945 10.3389/fcell.2026.1765945"],"submitter_affiliation":["Spanish National Center for Biotechnology"],"submitter_country":["Spain"],"doi":["10.6019/PXD072211"],"pubmed_abstract":["<h4>Background</h4>Epigenetic alterations play an increasingly recognized role in carcinogenesis and in the development of resistance to anticancer therapies. Epigenetic enzymes (writers and erasers) and effectors (readers) are largely influenced by the availability of metabolites generated through one-carbon metabolism (OCM), the tricarboxylic acid (TCA) cycle, and acetyl-CoA synthesis (ACS). In this study we examined the expression of epigenetic and metabolic genes to investigate their interplay in cholangiocarcinoma (CCA).<h4>Method</h4>We examined 257 epigenetic genes (EpiGs), 96 metabolic genes (MGs), and 189 rate-limiting enzymes (RLEs) in transcriptomic data from iCCA, eCCA, and normal bile ducts, healthy liver-derived organoids, and CCA tumoroids, alongside prognostic signatures. CRISPR-Cas9 DepMap data evaluated the impact of EpiGs and MGs on cell viability. HuCCT-1 iCCA cells were exposed to hypoxia (1% O<sub>2</sub>, 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (TAZ/Akt and NICD1/Akt) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in TAZ/Akt livers.<h4>Results</h4>Several EpiGs were upregulated in iCCA and eCCA, including writers (<i>DNMT1</i>, <i>EZH2</i>, <i>SUZ12</i>), readers (<i>CBX3</i>, <i>PHF20L1</i>, <i>SMARCA4</i>), and erasers (<i>HDAC1</i>, <i>HDAC3</i>, <i>KDM5C</i>). MGs in OCM, TCA, and ACS pathways were dysregulated (up: <i>GART</i>, <i>IDH2</i>, <i>TYMS</i>; down: <i>ALDH1L1</i>, <i>MAT1A</i>, <i>SHMT1</i>). Integrated analyses identified 27 EpiGs and 8 MGs whose overexpression predicted poor survival. Subsets of EpiGs, MGs, and RLEs were linked to proliferative, high-recurrence iCCA subclasses. CRISPR screens highlighted 50 EpiGs and 23 MGs essential for CCA viability. Tumor microenvironment (TME) analyses revealed distinct immune-stromal subclasses with coherent epigenetic-metabolic signatures. In CCA cells, hypoxia induced epigenetic programs that mirrored those in CCA patients. Transcriptomic analyses in human and multi-omic analyses (transcriptomic, metabolomic, and proteomic studies) in mouse CCA livers highlighted rewiring of nucleotide, one-carbon, lipid, and mitochondrial pathways, with evidence of metabolic-epigenetic crosstalk.<h4>Conclusion</h4>EpiGs and MGs are markedly altered in both human and experimental CCA, with several changes particularly enriched in aggressive molecular subclasses associated with poor prognosis. We observed substantial rewiring of epigenetic cofactor-related MG expression in CCAs. Functional assays validated new targets among EpiGs (e.g., <i>CBX3</i>, <i>CHD4</i>, <i>DEK</i>, <i>SMARCA4</i>, and <i>TRIM28</i>) and MGs (<i>TYMS</i> and <i>IDH2</i>) in CCA."],"pubmed_title":["Transcriptomic profiling of epigenetic regulators and metabolic reprogramming in human cholangiocarcinoma."],"pubmed_authors":["Lopez-Pascual Amaya A, Elurbide Jasmin J, Valbuena-Goiricelaya Emiliana E, Latasa M Ujue MU, Anaya Elena E, Adan-Villaescusa Elena E, Castelló-Uribe Borja B, Martínez-Pérez Luz A LA, Uriarte Iker I, Arechederra Maria M, Ciordia Sergio S, Corrales Fernando J FJ, Strnad Pavel P, Frankova Sona S, Sticova Eva E, Fabian Ondrej O, Colyn Leticia L, Inacio Patricia P, Bayo Juan J, Huch Meritxell M, Berasain Carmen C, Fernández-Barrena Maite G MG, Avila Matías A MA"],"additional_accession":[]},"is_claimable":false,"name":"Transcriptomic profiling of epigenetic regulators and metabolic reprogramming in human cholangiocarcinoma","description":"Background: Epigenetic alterations play an increasingly recognized role in carcinogenesis and in the development of resistance to anticancer therapies. Epigenetic enzymes (writers and erasers) and effectors (readers) are largely influenced by the availability of metabolites grenerated through one-carbon metabolism (OCM), the tricarboxylic acid (TCA) cycle, and acetyl-CoA synthesis (ACS). In this study we examined the expression of epigenetic and metabolic genes to investigate their interplay in cholangiocarcinoma (CCA).  Method: We examined 257 epigenetic genes (EpiGs), 96 metabolic genes (MGs), and 189 rate-limiting enzymes (RLEs) in transcriptomic data from iCCA, eCCA, CCA organoids, and normal bile ducts, alongside prognostic signatures. CRISPR-Cas9 DepMap data evaluated the impact of EpiGs and MGs on cell viability. HuCCT-1 iCCA cells were exposed to hypoxia (1% O₂, 24 h) to assess EpiG responses. Transcriptomic deconvolution characterized EpiGs, MGs, and RLEs expression across four tumor microenvironment (TME) subtypes. Two mouse CCA models (Akt/TAZ, Akt/NICD) underwent RNA-seq, complemented by multi-omic profiling (transcriptomic, proteomic, metabolomic) in Akt/TAZ livers.  Results: Several EpiGs were upregulated in iCCA and eCCA, including writers (DNMT1, EZH2, SUZ12), readers (CBX3, PHF20L1, SMARCA4), and erasers (HDAC1, HDAC3, KDM5C). MGs in OCM, TCA, and ACS pathways were dysregulated (up: GART, IDH2, TYMS; down: ALDH1L1, MAT1A, SHMT1). Integrated analyses identified 27 EpiGs and 8 MGs whose overexpression predicted poor survival. Subsets of EpiGs, MGs, and RLEs were linked to proliferative, high-recurrence iCCA subclasses. CRISPR screens highlighted 50 EpiGs and 23 MGs essential for CCA viability. Tumor microenvironment (TME) analyses revealed distinct immune-stromal subclasses with coherent epigenetic–metabolic signatures. In CCA cells, hypoxia induced epigenetic programs that mirrored those in CCA patients. Transcriptomic analyses in human and multi-omic analyses (transcriptomic, metabolomic, and proteomic studies) in mouse CCA livers highlighted rewiring of nucleotide, one-carbon, lipid, and mitochondrial pathways, with evidence of metabolic-epigenetic crosstalk.  Conclusion: EpiGs and MGs are markedly altered in both human and experimental CCA, with several changes particularly enriched in aggressive molecular subclasses associated with poor prognosis. We observed substantial rewiring of epigenetic cofactor-related MG expression in CCAs. Functional assays validated new targets among EpiGs (e.g. CBX3, CHD4, DEK, SMARCA4, and TRIM28) and MGs (TYMS and IDH2) in CCA.","dates":{"publication":"2026-06-29","submission":"2025-12-19"},"accession":"PXD072211","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:1073882","NEWT:6819","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:643680","NEWT:214092","NCBIT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