Summary of Study ST002572

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001659. The data can be accessed directly via it's Project DOI: 10.21228/M8872J This work is supported by NIH grant, U2C- DK119886.


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Study IDST002572
Study TitleSteady State and Cysteine Flux Metabolomics Study in PTEN WT and PTEN KO MEFs
Study TypeQuantitative Targeted Mass Spec
Study SummaryIn earlier studies we had indication that the tumor suppressor PTEN was downregulating the cystine glutamate antiporter, xCT; therefore to probe whether this effect on xCT was altering cystine uptake and downstream cystine metabolism, we performed steady state metabolomics via targeted LC-MS/MS on PTEN WT and PTEN KO MEFs. Steady state metabolomics revealed that Pten KO MEFs have a sixfold and fourfold increase in intracellular cystine and cysteine abundance, respectively, as well as a higher abundance of glutathione and the glutathione synthesis intermediate gamma-glutamylcysteine, compared to Pten WT MEFs. In addition to cystine import by xCT as a source of cysteine, cysteine can also be funneled into or recycled from the transsulfuration and choline oxidation pathways. Pten KO MEFs were also found to have increased abundance of transsulfuration pathway metabolites, as well as choline oxidation pathway metabolites. Collectively, this suggests that PTEN regulates cysteine and glutathione metabolism and that PTEN KO cells have more glutathione compared to PTEN WT cells. Next to determine if the increased glutathione in the Pten KO MEFs was being synthesized from increased cystine being brought into the cell by xCT, we performed cystine flux metabolomics via targeted LC-MS/MS on PTEN WT and PTEN KO MEFs and using the heavy isotope 13C2-cystine. Cystine Flux metabolomics revealed Pten KO MEFs were found to have a fourfold and threefold higher accumulation of 13C into intracellular cystine and cysteine, respectively, than Pten WT MEFs, indicating that more extracellular cystine is being brought into the cell by xCT. This result seems plausible given these cells were observed to have higher levels of xCT transporters compared to Pten WT MEFs. Furthermore, there was more cystine flux into glutathione synthesis in Pten KO MEFs, indicated by the sevenfold higher accumulation of heavy isotope labeled glutathione and higher accumulation of its preceding intermediate -glutamylcysteine. Together these findings suggest that PTEN loss heightened the cell’s ability to import cystine via xCT and as a result increased glutathione pools.
Mount Sinai
DepartmentOncological Sciences
LaboratoryRamon Parsons Laboratory
Last NameCahuzac
First NameKaitlyn
Address6358 Lucent Lane Sandy Springs GA 30328
Submit Date2023-04-20
Analysis Type DetailAPCI-MS
Release Date2023-04-23
Release Version1
Kaitlyn Cahuzac Kaitlyn Cahuzac application/zip

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Project ID:PR001659
Project DOI:doi: 10.21228/M8872J
Project Title:AKT activation due to PTEN loss upregulates xCT via the GSK3beta/NRF2 axis resulting in inhibition of ferroptosis and revealing a novel tumor suppressive property of PTEN
Project Type:Quantitative Targeted Mass Spec
Project Summary:Here we show that the tumor suppressor PTEN sensitizes cells to ferroptosis, an iron dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter, system Xc- (xCT), and augmenting cysteine metabolism. Loss of PTEN activated AKT kinase to inhibit GSK3beta, increasing NRF2 along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increased the flux of cystine transport and the synthesis of glutathione, which enhanced the steady state levels of these metabolites. A pan cancer analysis revealed that loss of PTEN shows evidence of increased xCT and PTEN mutant cells were found to be resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic stress that occurs during tumor initiation and progression.
Institute:Mount Sinai Oncological Sciences Department
Laboratory:Ramon Parsons Laboratory
Last Name:Cahuzac
First Name:Kaitlyn
Address:6358 Lucent Lane, Atlanta, GA, 30328, USA


Subject ID:SU002673
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Age Or Age Range:embryonic fibroblasts


Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA258362PTEN KO Rep3PTEN knock-out
SA258363PTEN KO Rep1PTEN knock-out
SA258364PTEN KO Rep2PTEN knock-out
SA258359PTEN WT Rep1PTEN Wild-Type
SA258360PTEN WT Rep3PTEN Wild-Type
SA258361PTEN WT Rep2PTEN Wild-Type
Showing results 1 to 6 of 6


Collection ID:CO002666
Collection Summary:PTEN WT and KO MEFs were seeded at 80% in 10cm plates 24 hours prior to metabolite extraction/sample prep
Sample Type:Fibroblasts


Treatment ID:TR002685
Treatment Summary:Cells did not undergo any treatment. They were cultured for 24 hours in 10cm plates with DMEM media containing 10% FBS, 1% Pen/strep, and an additional 1% of glutamine

Sample Preparation:

Sampleprep ID:SP002679
Sampleprep Summary:Media was fully removed from the plates. Metabolites were extracted using ice cold 80% methanol and dried using a speed vac. The dried metabolites were sent to the Mass Spec Core ran by John Asara at the Beth Israel Deaconess Medical Center at Harvard University for targeted LC-MS/MS. For detailed sample prep information see the attached protocol.

Combined analysis:

Analysis ID AN004237
Analysis type MS
Chromatography type HILIC
Chromatography system Waters Acquity
Column Waters XBridge Amide (100 x 4.6mm,3.5um)
MS instrument type QTRAP
MS instrument name ABI Sciex 5500 QTrap
Units peak area intensity


Chromatography ID:CH003144
Instrument Name:Waters Acquity
Column Name:Waters XBridge Amide (100 x 4.6mm,3.5um)
Column Temperature:45
Flow Gradient:85% buffer B to 30% buffer B from 0 - 3 minutes, then 30% buffer B to 3% buffer B from minute 3 to 12, then 2% buffer B held from minute 12 to 15, followed by 2% buffer B to 85% buffer B from minute 15 to 16, then 85% buffer B held from minute 16-23 in order to re-equilibrate the column.
Flow Rate:400uL/minute
Solvent A:95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate
Solvent B:100% acetonitrile
Chromatography Type:HILIC


MS ID:MS003984
Analysis ID:AN004237
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:QTRAP
MS Comments:Selective reaction monitoring, SRM, of endogenous water-soluble metabolites was performed for steady state analysis. The positive ion mode ESI voltage was +4900. Dwell time was set to 3ms per SRM transition and 1.55 seconds was the total cycle time. Retention time 15-20 seconds. MultiQuant v2.1 software was used to in integrate peak areas for each metabolite SRM transition