Summary of Study ST003256

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002015. The data can be accessed directly via it's Project DOI: 10.21228/M85238 This work is supported by NIH grant, U2C- DK119886.

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Study IDST003256
Study TitleExploration of Zeb1-dependent changes in the redox-lipidome of MDA-MB-231 cells
Study SummaryHuman breast cancer MDA-MB-231 wildtype (WT) cells and the stably transduced MDA-MB-231 shZeb1 (stable Zeb1 knockdown) and shCtrl cell lines (control cell line for the stable Zeb1 knockdown) (Spaderna et al. 2008, DOI: 10.1158/0008-5472.CAN-07-5682) were treated with DMSO or RSL3 (1 or 10 µM) for 2 h, 4 h, 6 h or 24 h. The cell pellets were collected and analyzed for their oxidized phospholipid profile by UPLC-MS/MS. Please note that one sample set was measured three times with the same sample-ID, but with different methods (Ox-PE, Ox-PC, Ox-PI), therefore each sub-class has their own raw-data file marked by their corresponding abbreviation (Ox-PE, Ox-PC, Ox-PI; e.g. "210514_MDA_ZEB1_oxPE_dil_UD_std_1ul_JZ_oxPE_MRM_003.wiff", "210514_MDA_ZEB1_oxPC_dil_UD_std_1ul_JZ_oxPC_MRM_002.wiff" or "210514_MDA_ZEB1_oxPI_dil_UD_std_1ul_JZ_oxPI_MRM_001.wiff").
Institute
University of Innsbruck
DepartmentMichael Popp Institute
Last NameKoeberle
First NameAndreas
AddressMitterweg 24, Innsbruck, Tyrol, 6020, Austria
EmailAndreas.Koeberle@uibk.ac.at
Phone+43 512 507 57903
Submit Date2024-05-27
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailLC-MS
Release Date2024-07-05
Release Version1
Andreas Koeberle Andreas Koeberle
https://dx.doi.org/10.21228/M85238
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002015
Project DOI:doi: 10.21228/M85238
Project Title:Zeb1-mediated control of the phospholipid PUFA/MUFA ratio in EMT/plasticity-associated 1 cancer cell ferroptosis
Project Summary:Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal-transition (EMT)-program. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical mediated peroxidation of phospholipids containing polyunsaturated fatty acids (PUFAs). We here describe that various forms of EMT-activation increase ferroptosis-susceptibility in cancer cells, which depends on the EMT-transcription factor Zeb1. To further investigate the underlying mechanisms of an EMT/Zeb1-coupled ferroptosis sensitivity, we analyzed key determinants of ferroptotic cell death, focusing on the proportion and (per)oxidation of fatty acid species in phospholipid subclasses. Using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we demonstrate that GPX4 inhibition in human breast cancer MDA-MB-231 cells (Zeb1high) led to a rapid (per)oxidation of PUFA-containing phospholipids (oxPL), which is absent in cells depleted of Zeb1 (shZeb1). Mechanistically, Zeb1 increases the ratio of phospholipids containing pro-ferroptotic PUFAs over cyto-protective monounsaturated fatty acids (MUFAs) in MDA-MB-231 cells, tumor-derived pancreatic cancer KPC cells as well as mice tumor allografts via the modulation of crucial lipogenic enzymes.
Institute:University of Innsbruck
Department:Michael Popp Institute
Last Name:Koeberle
First Name:Andreas
Address:Mitterweg 24, Innsbruck, Tyrol, 6020, Austria
Email:Andreas.Koeberle@uibk.ac.at
Phone:+43 512 507 57903
Funding Source:the Austrian Science Fund (FWF) (P 36299), the German Research Council (GRK 1715), and the Phospholipid Research Center (Grant Number AKO‐2019‐070/2‐1, AKO-2O22-100/2-2), the Tyrolean Science Fund (TWF) (F.33467/7-2021).
Publications:in revision
Contributors:Zhigang Rao, Jie Zhang, André Gollowitzer, Leonhard Bereuter, Andreas Koeberle

Subject:

Subject ID:SU003375
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Genotype Treatment Treatment Time
SA353986210514_MDA_ZEB1_n3_2h_sh-Ctrl_DMSO_22Breast cancer cells shCtrl DMSO 2 h
SA353987210514_MDA_ZEB1_n2_2h_sh-Ctrl_DMSO_13Breast cancer cells shCtrl DMSO 2 h
SA353988210514_MDA_ZEB1_n1_2h_sh-Ctrl_DMSO_4Breast cancer cells shCtrl DMSO 2 h
SA353992210514_MDA_ZEB1_n2_2h_sh-Ctrl_RSL3_10_uM_15Breast cancer cells shCtrl RSL3 (10 µM) 2 h
SA353993210514_MDA_ZEB1_n3_2h_sh-Ctrl_RSL3_10_uM_24Breast cancer cells shCtrl RSL3 (10 µM) 2 h
SA353994210514_MDA_ZEB1_n1_2h_sh-Ctrl_RSL3_10_uM_6Breast cancer cells shCtrl RSL3 (10 µM) 2 h
SA353989210514_MDA_ZEB1_n2_2h_sh-Ctrl_RSL3_1_uM_14Breast cancer cells shCtrl RSL3 (1 µM) 2 h
SA353990210514_MDA_ZEB1_n3_2h_sh-Ctrl_RSL3_1_uM_23Breast cancer cells shCtrl RSL3 (1 µM) 2 h
SA353991210514_MDA_ZEB1_n1_2h_sh-Ctrl_RSL3_1_uM_5Breast cancer cells shCtrl RSL3 (1 µM) 2 h
SA353995210514_MDA_ZEB1_n2_2h_sh-ZEB1_DMSO_16Breast cancer cells shZeb1 DMSO 2 h
SA353996210514_MDA_ZEB1_n1_2h_sh-ZEB1_DMSO_7Breast cancer cells shZeb1 DMSO 2 h
SA353997210514_MDA_ZEB1_n3_2h_sh-ZEB1_DMSO_25Breast cancer cells shZeb1 DMSO 2 h
SA354001210514_MDA_ZEB1_n3_2h_sh-ZEB1_RSL3_10_uM_27Breast cancer cells shZeb1 RSL3 (10 µM) 2 h
SA354002210514_MDA_ZEB1_n1_2h_sh-ZEB1_RSL3_10_uM_9Breast cancer cells shZeb1 RSL3 (10 µM) 2 h
SA354003210514_MDA_ZEB1_n2_2h_sh-ZEB1_RSL3_10_uM_18Breast cancer cells shZeb1 RSL3 (10 µM) 2 h
SA353998210514_MDA_ZEB1_n2_2h_sh-ZEB1_RSL3_1_uM_17Breast cancer cells shZeb1 RSL3 (1 µM) 2 h
SA353999210514_MDA_ZEB1_n3_2h_sh-ZEB1_RSL3_1_uM_26Breast cancer cells shZeb1 RSL3 (1 µM) 2 h
SA354000210514_MDA_ZEB1_n1_2h_sh-ZEB1_RSL3_1_uM_8Breast cancer cells shZeb1 RSL3 (1 µM) 2 h
SA353944210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_24h_DMSO_7Breast cancer cells WT DMSO 24 h
SA353945210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_24h_DMSO_77Breast cancer cells WT DMSO 24 h
SA353946210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_24h_DMSO_65Breast cancer cells WT DMSO 24 h
SA353947210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_24h_DMSO_46Breast cancer cells WT DMSO 24 h
SA353948210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_24h_DMSO_23Breast cancer cells WT DMSO 24 h
SA353935210514_MDA_ZEB1_n3_2h_WT_DMSO_19Breast cancer cells WT DMSO 2 h
SA353936210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_2h_DMSO_17_correctBreast cancer cells WT DMSO 2 h
SA353937210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_2h_RSL3_1uM_18Breast cancer cells WT DMSO 2 h
SA353938210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_2h_DMSO_73Breast cancer cells WT DMSO 2 h
SA353939210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_2h_DMSO_61Breast cancer cells WT DMSO 2 h
SA353940210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_2h_DMSO_37Breast cancer cells WT DMSO 2 h
SA353941210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_2h_DMSO_1Breast cancer cells WT DMSO 2 h
SA353942210514_MDA_ZEB1_n1_2h_WT_DMSO_1Breast cancer cells WT DMSO 2 h
SA353943210514_MDA_ZEB1_n2_2h_WT_DMSO_10Breast cancer cells WT DMSO 2 h
SA353949210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_4h_DMSO_19Breast cancer cells WT DMSO 4 h
SA353950210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_4h_DMSO_40Breast cancer cells WT DMSO 4 h
SA353951210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_4h_DMSO_3Breast cancer cells WT DMSO 4 h
SA353952210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_6h_DMSO_43Breast cancer cells WT DMSO 6 h
SA353953210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_6h_DMSO_21Breast cancer cells WT DMSO 6 h
SA353954210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_6h_DMSO_5Breast cancer cells WT DMSO 6 h
SA353977210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_24h_RSL3_10uM_48Breast cancer cells WT RSL3 (10 µM) 24 h
SA353978210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_24h_RSL3_10uM_66Breast cancer cells WT RSL3 (10 µM) 24 h
SA353979210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_24h_RSL3_10uM_78Breast cancer cells WT RSL3 (10 µM) 24 h
SA353971210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_2h_RSL3_10uM_62Breast cancer cells WT RSL3 (10 µM) 2 h
SA353972210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_2h_RSL3_10uM_39Breast cancer cells WT RSL3 (10 µM) 2 h
SA353973210514_MDA_ZEB1_n2_2h_WT_RSL3_10_uM_12Breast cancer cells WT RSL3 (10 µM) 2 h
SA353974210514_MDA_ZEB1_n3_2h_WT_RSL3_10_uM_21Breast cancer cells WT RSL3 (10 µM) 2 h
SA353975210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_2h_RSL3_10uM_74Breast cancer cells WT RSL3 (10 µM) 2 h
SA353976210514_MDA_ZEB1_n1_2h_WT_RSL3_10_uM_3Breast cancer cells WT RSL3 (10 µM) 2 h
SA353980210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_4h_RSL3_10uM_75Breast cancer cells WT RSL3 (10 µM) 4 h
SA353981210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_4h_RSL3_10uM_42Breast cancer cells WT RSL3 (10 µM) 4 h
SA353982210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_4h_RSL3_10uM_63Breast cancer cells WT RSL3 (10 µM) 4 h
SA353983210317_MDA_ZEB1_TC_pre_RSL3_WT_n5_6h_RSL3_10uM_76Breast cancer cells WT RSL3 (10 µM) 6 h
SA353984210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_6h_RSL3_10uM_45Breast cancer cells WT RSL3 (10 µM) 6 h
SA353985210317_MDA_ZEB1_TC_pre_RSL3_WT_n4_6h_RSL3_10uM_64Breast cancer cells WT RSL3 (10 µM) 6 h
SA353962210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_24h_RSL3_1uM_24Breast cancer cells WT RSL3 (1 µM) 24 h
SA353963210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_24h_RSL3_1uM_47Breast cancer cells WT RSL3 (1 µM) 24 h
SA353964210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_24h_RSL3_1uM_8Breast cancer cells WT RSL3 (1 µM) 24 h
SA353955210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_2h_RSL3_1uM_2Breast cancer cells WT RSL3 (1 µM) 2 h
SA353956210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_2h_RSL3_1uM_18_correctBreast cancer cells WT RSL3 (1 µM) 2 h
SA353957210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_2h_DMSO_17Breast cancer cells WT RSL3 (1 µM) 2 h
SA353958210514_MDA_ZEB1_n2_2h_WT_RSL3_1_uM_11Breast cancer cells WT RSL3 (1 µM) 2 h
SA353959210514_MDA_ZEB1_n1_2h_WT_RSL3_1_uM_2Breast cancer cells WT RSL3 (1 µM) 2 h
SA353960210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_2h_RSL3_1uM_38Breast cancer cells WT RSL3 (1 µM) 2 h
SA353961210514_MDA_ZEB1_n3_2h_WT_RSL3_1_uM_20Breast cancer cells WT RSL3 (1 µM) 2 h
SA353965210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_4h_RSL3_1uM_4Breast cancer cells WT RSL3 (1 µM) 4 h
SA353966210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_4h_RSL3_1uM_41Breast cancer cells WT RSL3 (1 µM) 4 h
SA353967210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_4h_RSL3_1uM_20Breast cancer cells WT RSL3 (1 µM) 4 h
SA353968210317_MDA_ZEB1_TC_pre_RSL3_WT_n3_6h_RSL3_1uM_44Breast cancer cells WT RSL3 (1 µM) 6 h
SA353969210317_MDA_ZEB1_TC_pre_RSL3_WT_n1_6h_RSL3_1uM_6Breast cancer cells WT RSL3 (1 µM) 6 h
SA353970210317_MDA_ZEB1_TC_pre_RSL3_WT_n2_6h_RSL3_1uM_22Breast cancer cells WT RSL3 (1 µM) 6 h
Showing results 1 to 69 of 69

Collection:

Collection ID:CO003368
Collection Summary:Cultured cells were washed, trypsinized, counted and flash-frozen in liquid N2 and stored at -80°C.
Sample Type:Breast cancer cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003384
Treatment Summary:Brest cancer cells (MDA-MB-231 cells) for oxPE, oxPC, oxPI: Human breast cancer MDA-MB-231 wildtype (WT) cells and the stably transduced MDA-MB-231 shZeb1 (stable Zeb1 knockdown) and shCtrl cell lines (control cell line for the stable Zeb1 knockdown) (Spaderna et al. 2008, DOI: 10.1158/0008-5472.CAN-07-5682) were treated with vehicle (DMSO) or RSL3 (1 and 10 µM) for 2 h, 4 h, 6 h, 24 h or 48 h at 37°C and 5% CO2.

Sample Preparation:

Sampleprep ID:SP003382
Sampleprep Summary:Phospholipids were extracted from cell pellets by successive addition of PBS pH 7.4, methanol, chloroform, and saline to a final ratio of 14:34:35:17. Evaporation of the organic layer yielded a lipid film that was dissolved in methanol and subjected to UPLC-MS/MS.
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005338
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity H-Class
Column Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex 6500+ QTrap
Ion Mode NEGATIVE
Units absolute intensities

Chromatography:

Chromatography ID:CH004040
Chromatography Summary:Chromatographic separation of phospholipids was carried out on an Acquity BEH C8 column (1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an Acquity UHPLC.
Instrument Name:Waters Acquity H-Class
Column Name:Waters ACQUITY UPLC BEH C8 (100 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:The gradient was ramped from 75 to 85% B over 5 min and further increased to 100% B within 2 min, followed by isocratic elution for another 2 min.
Flow Rate:0.75 mL/min
Solvent A:90% Water, 10% Acetonitrile; 2 mM ammonium acetate
Solvent B:5% Water, 95% Acetonitrile; 2 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS005068
Analysis ID:AN005338
Instrument Name:ABI Sciex 6500+ QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Targeted MRM with pre-optimized settings and subsequent automated integration of selected signals using Analyst 1.6.3 or Analyst 1.7.1 (Sciex). Oxidized phospholipid species were identified by the fragmentation of [M-H]- (Ox-PE, Ox-PI) or [M+OAc]- (Ox-PC) to the saturated fatty acid anion (16:0 and 18:0) and either the PUFA anion (20:4 and 22:4) with one to three oxygen incorporated or a secondary fragment. Oxidized phospholipids were quantified based on the most intensive, specific transition to the oxidized fatty acid anions.
Ion Mode:NEGATIVE
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