#METABOLOMICS WORKBENCH akoeberle_20240527_105303 DATATRACK_ID:4865 STUDY_ID:ST003259 ANALYSIS_ID:AN005343 PROJECT_ID:PR002015 VERSION 1 CREATED_ON May 27, 2024, 11:18 am #PROJECT PR:PROJECT_TITLE Zeb1-mediated control of the phospholipid PUFA/MUFA ratio in PR:PROJECT_TITLE EMT/plasticity-associated 1 cancer cell ferroptosis PR:PROJECT_SUMMARY Therapy resistance and metastasis, the most fatal steps in cancer, are often PR:PROJECT_SUMMARY triggered by a (partial) activation of the epithelial-mesenchymal-transition PR:PROJECT_SUMMARY (EMT)-program. A mesenchymal phenotype predisposes to ferroptosis, a cell death PR:PROJECT_SUMMARY pathway exerted by an iron and oxygen-radical mediated peroxidation of PR:PROJECT_SUMMARY phospholipids containing polyunsaturated fatty acids (PUFAs). We here describe PR:PROJECT_SUMMARY that various forms of EMT-activation increase ferroptosis-susceptibility in PR:PROJECT_SUMMARY cancer cells, which depends on the EMT-transcription factor Zeb1. To further PR:PROJECT_SUMMARY investigate the underlying mechanisms of an EMT/Zeb1-coupled ferroptosis PR:PROJECT_SUMMARY sensitivity, we analyzed key determinants of ferroptotic cell death, focusing on PR:PROJECT_SUMMARY the proportion and (per)oxidation of fatty acid species in phospholipid PR:PROJECT_SUMMARY subclasses. Using ultra-performance liquid chromatography-tandem mass PR:PROJECT_SUMMARY spectrometry (UPLC-MS/MS), we demonstrate that GPX4 inhibition in human breast PR:PROJECT_SUMMARY cancer MDA-MB-231 cells (Zeb1high) led to a rapid (per)oxidation of PR:PROJECT_SUMMARY PUFA-containing phospholipids (oxPL), which is absent in cells depleted of Zeb1 PR:PROJECT_SUMMARY (shZeb1). Mechanistically, Zeb1 increases the ratio of phospholipids containing PR:PROJECT_SUMMARY pro-ferroptotic PUFAs over cyto-protective monounsaturated fatty acids (MUFAs) PR:PROJECT_SUMMARY in MDA-MB-231 cells, tumor-derived pancreatic cancer KPC cells as well as mice PR:PROJECT_SUMMARY tumor allografts via the modulation of crucial lipogenic enzymes. PR:INSTITUTE University of Innsbruck PR:DEPARTMENT Michael Popp Institute PR:LAST_NAME Koeberle PR:FIRST_NAME Andreas PR:ADDRESS Mitterweg 24, Innsbruck, Tyrol, 6020, Austria PR:EMAIL Andreas.Koeberle@uibk.ac.at PR:PHONE +43 512 507 57903 PR:FUNDING_SOURCE the Austrian Science Fund (FWF) (P 36299), the German Research Council (GRK PR:FUNDING_SOURCE 1715), and the Phospholipid Research Center (Grant Number PR:FUNDING_SOURCE AKO‐2019‐070/2‐1, AKO-2O22-100/2-2), the Tyrolean Science Fund (TWF) PR:FUNDING_SOURCE (F.33467/7-2021). PR:PUBLICATIONS in revision PR:CONTRIBUTORS Zhigang Rao, Jie Zhang, André Gollowitzer, Leonhard Bereuter, Andreas Koeberle #STUDY ST:STUDY_TITLE Exploration of EMT-dependent changes of phosphatidylethanolamine profiles in KPC ST:STUDY_TITLE allografts ST:STUDY_SUMMARY Cryo-conserved tumors from subcutaneous allografts as described (Krebs et al. ST:STUDY_SUMMARY 2017, DOI: 10.1038/ncb3513) were retrieved. Three tumors derived from ST:STUDY_SUMMARY mesenchymal KPC cell lines (lines KPC550 and KPC701) and epithelial KPC cell ST:STUDY_SUMMARY lines (lines KPC438 and KPC661) were analyzed for their phosphatidylethanolamine ST:STUDY_SUMMARY profile by UPLC-MS/MS. ST:INSTITUTE University of Innsbruck ST:DEPARTMENT Michael Popp Institute ST:LAST_NAME Koeberle ST:FIRST_NAME Andreas ST:ADDRESS Mitterweg 24, Innsbruck, Tyrol, 6020, Austria ST:EMAIL Andreas.Koeberle@uibk.ac.at ST:PHONE +43 512 507 57903 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS KPC661 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_01 Sample source:KPC allografts | Phenotype:epithelial/mixed RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff SUBJECT_SAMPLE_FACTORS KPC661 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_02 Sample source:KPC allografts | Phenotype:epithelial/mixed RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff SUBJECT_SAMPLE_FACTORS KPC438 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_03 Sample source:KPC allografts | Phenotype:epithelial/mixed RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff SUBJECT_SAMPLE_FACTORS KPC701 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_04 Sample source:KPC allografts | Phenotype:mesenchymal RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff SUBJECT_SAMPLE_FACTORS KPC550 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_05 Sample source:KPC allografts | Phenotype:mesenchymal RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff SUBJECT_SAMPLE_FACTORS KPC550 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_06 Sample source:KPC allografts | Phenotype:mesenchymal RAW_FILE_NAME(Raw file name (Analyst file name))=230221_PE_KPC-Brabletz_1-30-dil_.wiff #COLLECTION CO:COLLECTION_SUMMARY Cryo-conserved tumors from subcutaneous allografts described in (Krebs et al. CO:COLLECTION_SUMMARY 2017, DOI: 10.1038/ncb3513) were retrieved. CO:SAMPLE_TYPE Tumor allograft CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY KPC cells were subcutaneously injected into the flanks of C57BL/6 mice for TR:TREATMENT_SUMMARY engraftment as described (Krebs et al. 2017, DOI: 10.1038/ncb3513). Three tumor TR:TREATMENT_SUMMARY allografts derived from mesenchymal KPC cell lines (lines KPC550 and KPC701) and TR:TREATMENT_SUMMARY epithelial KPC cell lines (lines KPC438 and KPC661) were collected, TR:TREATMENT_SUMMARY cryo-conserved and stored at -80°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Phospholipids were extracted from allograft tumor tissue by successive addition SP:SAMPLEPREP_SUMMARY of PBS pH 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. SP:SAMPLEPREP_SUMMARY Evaporation of the organic layer yielded a lipid film that was dissolved in SP:SAMPLEPREP_SUMMARY methanol and subjected to UPLC-MS/MS. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 CH:CHROMATOGRAPHY_SUMMARY column (1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an Acquity UHPLC. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity H-Class CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um) CH:SOLVENT_A water/acetonitrile 90/10, 2 mM ammonium acetate CH:SOLVENT_B water/acetonitrile 5/95, 2 mM ammonium acetate CH:FLOW_GRADIENT The gradient was ramped from 75 to 85% B over 5 min and further increased to CH:FLOW_GRADIENT 100% B within 2 min, followed by isocratic elution for another 2 min. CH:FLOW_RATE 0.75 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Targeted MRM with pre-optimized settings and subsequent automated integration of MS:MS_COMMENTS selected signals using Analyst 1.6.3 or Analyst 1.7.1 (Sciex). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative intensities MS_METABOLITE_DATA_START Samples 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_01 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_02 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_03 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_04 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_05 230221_PE_KPC-Brabletz_1-30-dil_Sample-1-24_06 Factors Sample source:KPC allografts | Phenotype:epithelial/mixed Sample source:KPC allografts | Phenotype:epithelial/mixed Sample source:KPC allografts | Phenotype:epithelial/mixed Sample source:KPC allografts | Phenotype:mesenchymal Sample source:KPC allografts | Phenotype:mesenchymal Sample source:KPC allografts | Phenotype:mesenchymal PE 14:0_16:0 0.0267275951 0.0407931480 0.0665213963 0.0547799102 0.0444761201 0.0857394433 PE 16:0/16:0 0.1600781989 0.1706996434 0.3178626739 0.2521749731 0.2306752654 0.4494248188 PE 16:0_16:1 0.2885058007 0.3856817697 0.4066306823 0.4896299015 0.3009908905 0.6038978939 PE 16:1_18:0 0.6020019029 0.5775701434 0.6517087497 0.8960915507 0.3218924817 0.7601011771 PE 16:0_18:0 0.6969069712 0.7237921089 0.9066089981 0.8935420389 0.5941183424 0.9592831430 PE 14:0_18:1 0.0782877386 0.1183663285 0.1199729366 0.1160359040 0.0636926888 0.0913540604 PE 16:0_18:1 4.1592961053 4.5082153866 5.2584478786 3.1195542965 2.1008874344 2.7773604233 PE 16:1_18:1 0.4571373325 0.6887914090 0.5007447580 0.6406973038 0.2513491958 0.5592956600 PE 18:0_18:1 36.7208369708 37.2108073323 29.4787741108 20.0328546981 8.1413426155 10.1445426162 PE 18:1/18:1 8.9417571438 6.6191087146 5.4253298164 4.7532054608 2.3830771956 3.2708963287 PE 16:0_18:2 0.9579561967 1.8731596285 1.8409575708 1.5244505131 2.1114778177 1.7580295823 PE 18:0_18:2 6.4453982512 6.8924572476 7.7247453916 9.1021543352 9.9016012475 8.1882159233 PE 18:1_18:2 1.3037339083 2.2082959146 1.7345948814 1.8555985257 1.5447585401 1.5565196045 PE 18:1_20:1 3.2146012169 2.6294180735 0.9540644794 0.6775121355 0.2654907558 0.3308529011 PE 18:0_20:2 0.9300097830 0.8203985135 0.5724645655 0.7635265085 0.4780852640 0.3992362778 PE 16:0_20:3 0.2110073300 0.2379600299 0.2646394680 0.2462339631 0.2228882677 0.3774170909 PE 18:0_20:3 4.1632032577 3.5455404819 3.7801940351 6.7559529130 3.3312064771 5.8063726207 PE 18:1_20:3 0.2848016173 0.2987400312 0.2959852024 0.3613770964 0.2042300497 0.4891721997 PE 16:0_20:4 1.1288892155 1.7399708950 2.3533656455 1.9729481938 2.8637307065 3.9697528830 PE 16:1_20:4 0.0502539076 0.0569082306 0.0821455522 0.0914850776 0.0819246660 0.1919161456 PE 18:0_20:4 14.0230397296 11.1609471378 17.9791883625 19.0886131453 22.8039193857 28.3729995038 PE 18:1_20:4 0.9349634313 1.4018171683 1.6044291011 1.7156212051 1.7830662596 3.0680248325 PE 16:0_22:4 0.4536657595 0.4830767523 1.0373988922 0.6513619762 1.1299953929 1.1554471204 PE 18:0_22:4 7.6909725106 5.2595039559 7.4577890915 10.3168303404 10.6496997415 8.9324226251 PE 18:1_22:4 0.3692628275 0.3493360589 0.6796751363 0.6721584553 0.8477092497 0.9251846146 PE 16:0_22:5 0.1742739682 0.3170790560 0.3500243229 0.3635851031 1.0402604646 0.6524490577 PE 18:0_22:5 1.2995177809 2.1378955293 1.4313730036 2.9061136489 7.7342468194 3.1460501027 PE 18:1_22:5 0.0929437049 0.1739970895 0.1726207401 0.2835492763 0.4530417411 0.3563491092 PE 16:0_22:6 0.4854454730 1.0481226837 1.2248278736 1.3203880060 3.4149125475 2.0804631912 PE 18:0_22:6 1.1756122672 2.9566086416 2.0550088107 3.7672340535 10.4013658055 4.3508733301 PE 18:1_22:6 0.2072443660 0.8420340898 0.5932962659 0.8955881251 2.2756345749 1.4509439980 LPE 16:0 0.4306840469 0.4907952453 0.4789357867 0.5991058176 0.5018508951 0.7307956146 LPE 16:1 0.0156748302 0.0232592510 0.0243556600 0.0493121496 0.0148394983 0.0288367999 LPE 18:0 1.2239831856 1.2597031445 1.3991413321 1.9555726531 1.0909606991 1.4859642505 LPE 18:1 0.4470140046 0.5384337698 0.4762749309 0.5284496033 0.2592342372 0.3239202184 LPE 18:2 0.0691099246 0.0929296538 0.0882588228 0.0914939096 0.0723795284 0.0543237908 LPE 20:4 0.0721243150 0.0991917598 0.1946626124 0.1722245225 0.0636453287 0.0994145386 LPE 22:5 0.0074736787 0.0086130796 0.0084909920 0.0109517132 0.0148394983 0.0083218528 LPE 22:6 0.0056037518 0.0099809025 0.0084894697 0.0120409965 0.0105023101 0.0078346557 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name metaboite name (ID in Analyst file) formula exactmass super_class main_class sub_class PE 14:0_16:0 14:0/16:0 C35H70NO8P 663.4839 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0/16:0 16:0/16:0 C37H74NO8P 691.5152 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_16:1 16:0/16:1 C37H72NO8P 689.4996 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:1_18:0 18:0/16:1 C39H76NO8P 717.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_18:0 16:0/18:0 C39H78NO8P 719.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 14:0_18:1 14:0/18:1 C37H72NO8P 689.4996 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_18:1 16:0/18:1 C39H76NO8P 717.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:1_18:1 16:1/18:1 C39H74NO8P 715.5152 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_18:1 18:0/18:1 C41H80NO8P 745.5622 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1/18:1 18:1/18:1 C41H78NO8P 743.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_18:2 16:0/18:2 C39H74NO8P 715.5152 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_18:2 18:0/18:2 C41H78NO8P 743.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_18:2 18:1/18:2 C41H76NO8P 741.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_20:1 18:1/20:1 C43H82NO8P 771.5778 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_20:2 18:0/20:2 C43H82NO8P 771.5778 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_20:3 16:0/20:3 C41H76NO8P 741.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_20:3 18:0/20:3 C43H80NO8P 769.5622 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_20:3 18:1/20:3 C43H78NO8P 767.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_20:4 16:0/20:4 C41H74NO8P 739.5152 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:1_20:4 16:1/20:4 C41H72NO8P 737.4996 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_20:4 18:0/20:4 C43H78NO8P 767.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_20:4 18:1/20:4 C43H76NO8P 765.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_22:4 16:0/22:4 C43H78NO8P 767.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_22:4 18:0/22:4 C45H82NO8P 795.5778 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_22:4 18:1/22:4 C45H80NO8P 793.5622 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_22:5 16:0/22:5 C43H76NO8P 765.5309 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_22:5 18:0/22:5 C45H80NO8P 793.5622 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_22:5 18:1/22:5 C45H78NO8P 791.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 16:0_22:6 16:0/22:6 C43H74NO8P 763.5152 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:0_22:6 18:0/22:6 C45H78NO8P 791.5465 Glycerophospholipids Glycerophosphoethanolamines PE PE 18:1_22:6 18:1/22:6 C45H76NO8P 789.5309 Glycerophospholipids Glycerophosphoethanolamines PE LPE 16:0 16:0-LPL C21H44NO7P 453.2855 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 16:1 16:1-LPL C21H42NO7P 451.2699 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 18:0 18:0-LPL C23H48NO7P 481.3168 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 18:1 18:1-LPL C23H46NO7P 479.3012 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 18:2 18:2-LPL C23H44NO7P 477.2855 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 20:4 20:4-LPL C25H44NO7P 501.2855 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 22:5 22:5-LPL C27H46NO7P 527.3012 Glycerophospholipids Glycerophosphoethanolamines LPE LPE 22:6 22:6-LPL C27H44NO7P 525.2855 Glycerophospholipids Glycerophosphoethanolamines LPE METABOLITES_END #END