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Conditioned macrophage supernatant was then collected, filtered (0.22 µm) and added to L513 cells (50% v/v) in 6-well plates for 6 h. Then, cells were treated to treatment for 16 h with 1μM 5-FU or DMSO (vehicle). After treatment, cells were washed with PBS and lysed with 1% SDS, 8M urea, Tris buffer pH 8.0 plus protease inhibitor. The cell lysates were subjected to 1min sonication using Branson probe sonicator with 3s on/off pulses and a 30% amplitude. The concentration of proteins was measured using BCA assay and the volume corresponding to 25 µg of protein was transferred from each sample to new tubes. Dithiothreitol (DTT) was added to a final concentration of 10mM, and samples were incubated for 45min at room temperature. Subsequently, iodoacetamide (IAA) was added to a final concentration of 50mM and samples were incubated at room temperature for 1h in the dark. The reaction was quenched by adding an additional 10mM of DTT. Proteins were precipitated by methanol/chloroform and resuspended in 20mM EPPS buffer pH 8.5 with 8M urea. Urea was diluted to 4M by adding 20 mM EPPS. Lysyl endopeptidase (LysC; Wako) was added at a 1:75 w/w ratio at room temperature overnight. Samples were diluted with 20 mM EPPS to the final urea concentration of 1M, and trypsin was added at a 1:75 w/w ratio, followed by incubation for 6 h at room temperature.  Acetonitrile (ACN) was added to a final concentration of 20% and TMTpro-16 reagents were added 4x by weight (200μg) to each sample, followed by incubation for 2h at room temperature. The reaction was quenched by addition of 0.5% hydroxylamine. Samples were pooled, acidified by TFA, cleaned using Sep-Pak C18 cartridges (Waters) and dried using DNA 120 SpeedVac Concentrator (Thermo). The TMT multiplexing scheme starting from TMT126 is four replicates of control (DMSO, no supernatant), four replicates of cells treated with supernatant (plus DMSO), four replicates of cells treated with 5-FU alone and four replicates of the combination.  The pooled sample was fractionated off-line by capillary reversed-phase chromatography at pH 10 into 24 fractions and dried. LC-MS/MS analyses were performed on a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientiﬁc). The instrument was equipped with an EASY ElectroSpray source and connected online to an Ultimate 3000 nanoflow UPLC system. The samples were pre-concentrated and desalted  online using a PepMap C18 nano-trap column (length - 2 cm; inner diameter - 75 µm; particle size - 3 µm; pore size - 100 Å; Thermo Fisher Scientiﬁc) with a ﬂow rate of 3 µL/min for 5 min. Peptide separation was performed on an EASY-Spray C18 reversed- phase nano-LC column (Acclaim PepMap RSLC; length - 50 cm; inner diameter - 2 µm; particle size - 2 µm; pore size – 100 Å; Thermo Scientiﬁc) at 55 °C and a ﬂow rate of 300 nL/min. Peptides were separated using a binary solvent system consisting of 0.1% (v/v) formic acid (FA), 2% (v/v) acetonitrile (ACN) as solvent A and 98% ACN (v/v), 0.1% (v/v) FA as solvent B. They were eluted with a gradient of 3–26% B in 97 min, and 26–95% B in 9 min. Subsequently, the analytical column was washed with 95% B for 5 min before re-equilibration with 3% B. The mass spectrometer was operated in a data-dependent acquisition mode. A survey mass spectrum (from m/z 375 to 1700) was acquired in the Orbitrap analyzer at a nominal resolution of 120,000. The automatic gain control (AGC) target was set 3x106, with the maximum injection time of 100 ms. The 18 most abundant ions in charge states 2+ to 7+ were isolated with 1.4 Th, fragmented using HCD MS/MS with 34% normalized collision energy, and detected in the Orbitrap analyzer at a nominal mass resolution of 60,000. The AGC target for MS/MS was set 5x105 with a maximum injection time of 54 ms, whereas dynamic exclusion was set to 30 s."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Proteome Discoverer 3.0 software (Thermo Scientific) with MS Amanda 2.0 search engine was utilized for the database search and quantification against the SwissProt Homo sapiens protein database (version 2025_02_05; 20,340 entries) . Cysteine carbamidomethylation was set as a fixed modification, along with TMTpro modifications (+304.207 Da), methionine oxidation, deamidation of arginine and asparagine as variable modifications. Enzyme specificity was defined as trypsin with a maximum of two missed cleavages. A 1% false discovery rate was employed as a filter at both the protein and peptide levels using Percolator. Contaminants were removed, and proteins with missing values were eliminated. The quantified abundance of each protein in each sample (labeled with a different TMTpro reagent) was normalized to the total intensity of all proteins in that sample. For each protein, the average normalized protein abundance in the treated replicates was divided by the average normalized abundance of that protein in the vehicle-treated replicates. The average ratio across replicates of each compound compared to the vehicle control was calculated, and the Log2 values of these ratios were determined. Two-tailed Student's t-test was employed to calculate the p-value."],"omics_type":["Proteomics"],"labhead":["Akos Vegvari"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Karolinska Institutet, Stockholm, Sweden"],"species":["Homo Sapiens (human)"],"publication":["42002547 Fragkoulis K, Łasut-Szyszka B, Végvári Á, Ayona D, Saei AA, Aschtgen MS, Peuget S. Klebsiella pneumoniae LPS drives stromal-mediated repression of p53 and colorectal cancer chemoresistance. Cell Death Dis. 2026 17(1):395 10.1038/s41419-026-08756-4"],"submitter_mail":["akos.vegvari@ki.se"],"submitter_affiliation":["Karolinska Institutet"],"submitter_country":["Sweden"],"pubmed_abstract":["The gut bacterial microbiota is increasingly recognized as a key modulator of colorectal cancer (CRC) initiation, progression and response to therapy. However, the mechanisms by which bacteria influence the response to anticancer drugs remain poorly understood. Here, we investigate the effects of microbiota-driven signaling on the tumor suppressor p53 and its impact on chemotherapy. We uncover a mechanism by which lipopolysaccharide (LPS) from Klebsiella pneumoniae and other Enterobacteria impairs p53 activity and promotes chemoresistance via paracrine signaling from the tumor microenvironment. While direct exposure to LPS did not alter the drug response of CRC cells, conditioned media from LPS-stimulated macrophages or fibroblasts suppressed p53 accumulation and attenuated the response to chemotherapeutic agents. Deep quantitative proteomics further revealed selective inhibition of a subset of p53 targets by inflammation. This same subset negatively correlated with inflammatory signature and immune infiltration in patients and was associated with improved survival following chemotherapy. Mechanistically, our data suggest that macrophage-derived extracellular vesicles contribute to p53 degradation in cancer cells. Overall, our findings reveal a microbiota-driven mechanism of p53 suppression via the microenvironment that contributes to chemoresistance, highlighting the impact of bacteria on tumor cell fate and therapeutic efficacy in CRC."],"pubmed_title":["Klebsiella pneumoniae LPS drives stromal-mediated repression of p53 and colorectal cancer chemoresistance."],"pubmed_authors":["Fragkoulis Konstantinos K, Łasut-Szyszka Barbara B, Végvári Ákos Á, Ayona Diyoly D, Saei Amir Ata AA, Aschtgen Marie-Stéphanie MS, Peuget Sylvain S"],"additional_accession":[]},"is_claimable":false,"name":"Klebsiella pneumoniae LPS drives stromal-mediated repression of p53 and colorectal cancer chemoresistance","description":"Evidence has emerged that the bacterial microbiota is a key modulator of colorectal cancer (CRC) response to therapy, but the molecular mechanisms of this interplay are poorly understood. Here, we investigate the effects of microbiota-driven signalling on the tumour suppressor p53 and its impact on chemotherapy. We uncover a mechanism by which lipopolysaccharide (LPS) from Klebsiella pneumoniae and other Enterobacteria impairs p53 activity and promotes chemoresistance via paracrine signalling from the tumour microenvironment. We observed that conditioned media from LPS-stimulated macrophages or fibroblasts suppressed p53 accumulation and attenuated the response to chemotherapeutic agents. Our data revealed selective inhibition of a subset of p53 targets by inflammation. This same subset negatively correlated with inflammatory signature and immune infiltration in patients and was associated with improved survival following chemotherapy. Our findings reveal a microbiota-driven mechanism of p53 suppression via the microenvironment that contributes to chemoresistance and highlight the impact of bacteria on tumour cell fate and therapeutic efficacy in CRC.","dates":{"publication":"2026-04-27","submission":"2025-06-02"},"accession":"PXD064521","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:1310173","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:460523","NEWT:2423","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:941638","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:300641","NEWT:39655","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:280406","NEWT:83906","NEWT:99488","NEWT:99487","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:9938","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:483179","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:11294","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT:4233","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:523840","NEWT:1076878","NEWT:200308","NEWT:200302","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:1170227","NEWT:2666255","NEWT:999810","NEWT:28995","NEWT:1392998","NEWT:1150621","NEWT:1085791","NEWT:745531","NEWT:226900","NEWT:231490","NEWT:590651","NEWT:449239","NEWT:244704","NEWT:9909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