#METABOLOMICS WORKBENCH kaitlyncahuzac_20230420_184108 DATATRACK_ID:3877 STUDY_ID:ST002572 ANALYSIS_ID:AN004237 PROJECT_ID:PR001659 VERSION 1 CREATED_ON April 21, 2023, 2:07 pm #PROJECT PR:PROJECT_TITLE AKT activation due to PTEN loss upregulates xCT via the GSK3beta/NRF2 axis PR:PROJECT_TITLE resulting in inhibition of ferroptosis and revealing a novel tumor suppressive PR:PROJECT_TITLE property of PTEN PR:PROJECT_TYPE Quantitative Targeted Mass Spec PR:PROJECT_SUMMARY Here we show that the tumor suppressor PTEN sensitizes cells to ferroptosis, an PR:PROJECT_SUMMARY iron dependent form of cell death, by restraining the expression and activity of PR:PROJECT_SUMMARY the cystine/glutamate antiporter, system Xc- (xCT), and augmenting cysteine PR:PROJECT_SUMMARY metabolism. Loss of PTEN activated AKT kinase to inhibit GSK3beta, increasing PR:PROJECT_SUMMARY NRF2 along with transcription of one of its known target genes encoding xCT. PR:PROJECT_SUMMARY Elevated xCT in Pten-null mouse embryonic fibroblasts increased the flux of PR:PROJECT_SUMMARY cystine transport and the synthesis of glutathione, which enhanced the steady PR:PROJECT_SUMMARY state levels of these metabolites. A pan cancer analysis revealed that loss of PR:PROJECT_SUMMARY PTEN shows evidence of increased xCT and PTEN mutant cells were found to be PR:PROJECT_SUMMARY resistant to ferroptosis as a consequence of elevated xCT. These findings PR:PROJECT_SUMMARY suggest that selection of PTEN mutation during tumor development may be due to PR:PROJECT_SUMMARY its ability to confer resistance to ferroptosis in the setting of metabolic PR:PROJECT_SUMMARY stress that occurs during tumor initiation and progression. PR:INSTITUTE Mount Sinai Oncological Sciences Department PR:LABORATORY Ramon Parsons Laboratory PR:LAST_NAME Cahuzac PR:FIRST_NAME Kaitlyn PR:ADDRESS 6358 Lucent Lane, Atlanta, GA, 30328, USA PR:EMAIL kaitlyncahuzac@gmail.com PR:PHONE 6784537911 #STUDY ST:STUDY_TITLE Steady State and Cysteine Flux Metabolomics Study in PTEN WT and PTEN KO MEFs ST:STUDY_TYPE Quantitative Targeted Mass Spec ST:STUDY_SUMMARY In earlier studies we had indication that the tumor suppressor PTEN was ST:STUDY_SUMMARY downregulating the cystine glutamate antiporter, xCT; therefore to probe whether ST:STUDY_SUMMARY this effect on xCT was altering cystine uptake and downstream cystine ST:STUDY_SUMMARY metabolism, we performed steady state metabolomics via targeted LC-MS/MS on PTEN ST:STUDY_SUMMARY WT and PTEN KO MEFs. Steady state metabolomics revealed that Pten KO MEFs have a ST:STUDY_SUMMARY sixfold and fourfold increase in intracellular cystine and cysteine abundance, ST:STUDY_SUMMARY respectively, as well as a higher abundance of glutathione and the glutathione ST:STUDY_SUMMARY synthesis intermediate gamma-glutamylcysteine, compared to Pten WT MEFs. In ST:STUDY_SUMMARY addition to cystine import by xCT as a source of cysteine, cysteine can also be ST:STUDY_SUMMARY funneled into or recycled from the transsulfuration and choline oxidation ST:STUDY_SUMMARY pathways. Pten KO MEFs were also found to have increased abundance of ST:STUDY_SUMMARY transsulfuration pathway metabolites, as well as choline oxidation pathway ST:STUDY_SUMMARY metabolites. Collectively, this suggests that PTEN regulates cysteine and ST:STUDY_SUMMARY glutathione metabolism and that PTEN KO cells have more glutathione compared to ST:STUDY_SUMMARY PTEN WT cells. Next to determine if the increased glutathione in the Pten KO ST:STUDY_SUMMARY MEFs was being synthesized from increased cystine being brought into the cell by ST:STUDY_SUMMARY xCT, we performed cystine flux metabolomics via targeted LC-MS/MS on PTEN WT and ST:STUDY_SUMMARY PTEN KO MEFs and using the heavy isotope 13C2-cystine. Cystine Flux metabolomics ST:STUDY_SUMMARY revealed Pten KO MEFs were found to have a fourfold and threefold higher ST:STUDY_SUMMARY accumulation of 13C into intracellular cystine and cysteine, respectively, than ST:STUDY_SUMMARY Pten WT MEFs, indicating that more extracellular cystine is being brought into ST:STUDY_SUMMARY the cell by xCT. This result seems plausible given these cells were observed to ST:STUDY_SUMMARY have higher levels of xCT transporters compared to Pten WT MEFs. Furthermore, ST:STUDY_SUMMARY there was more cystine flux into glutathione synthesis in Pten KO MEFs, ST:STUDY_SUMMARY indicated by the sevenfold higher accumulation of heavy isotope labeled ST:STUDY_SUMMARY glutathione and higher accumulation of its preceding intermediate ST:STUDY_SUMMARY -glutamylcysteine. Together these findings suggest that PTEN loss heightened ST:STUDY_SUMMARY the cell’s ability to import cystine via xCT and as a result increased ST:STUDY_SUMMARY glutathione pools. ST:INSTITUTE Mount Sinai ST:DEPARTMENT Oncological Sciences ST:LABORATORY Ramon Parsons Laboratory ST:LAST_NAME Cahuzac ST:FIRST_NAME Kaitlyn ST:ADDRESS 6358 Lucent Lane Sandy Springs GA 30328 ST:EMAIL kaitlyncahuzac@gmail.com ST:PHONE 6784537911 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE embryonic fibroblasts #FACTORS #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 - PTEN WT Rep1 Genotype:PTEN Wild-Type SUBJECT_SAMPLE_FACTORS - PTEN WT Rep2 Genotype:PTEN Wild-Type SUBJECT_SAMPLE_FACTORS - PTEN WT Rep3 Genotype:PTEN Wild-Type SUBJECT_SAMPLE_FACTORS - PTEN KO Rep1 Genotype:PTEN knock-out SUBJECT_SAMPLE_FACTORS - PTEN KO Rep2 Genotype:PTEN knock-out SUBJECT_SAMPLE_FACTORS - PTEN KO Rep3 Genotype:PTEN knock-out #COLLECTION CO:COLLECTION_SUMMARY PTEN WT and KO MEFs were seeded at 80% in 10cm plates 24 hours prior to CO:COLLECTION_SUMMARY metabolite extraction/sample prep CO:SAMPLE_TYPE Fibroblasts #TREATMENT TR:TREATMENT_SUMMARY Cells did not undergo any treatment. They were cultured for 24 hours in 10cm TR:TREATMENT_SUMMARY plates with DMEM media containing 10% FBS, 1% Pen/strep, and an additional 1% of TR:TREATMENT_SUMMARY glutamine #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Media was fully removed from the plates. Metabolites were extracted using ice SP:SAMPLEPREP_SUMMARY cold 80% methanol and dried using a speed vac. The dried metabolites were sent SP:SAMPLEPREP_SUMMARY to the Mass Spec Core ran by John Asara at the Beth Israel Deaconess Medical SP:SAMPLEPREP_SUMMARY Center at Harvard University for targeted LC-MS/MS. For detailed sample prep SP:SAMPLEPREP_SUMMARY information see the attached protocol. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm,3.5um) CH:SOLVENT_A 95% water/5% acetonitrile; 20 mM ammonium hydroxide; 20 mM ammonium acetate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 85% buffer B to 30% buffer B from 0 - 3 minutes, then 30% buffer B to 3% buffer CH:FLOW_GRADIENT B from minute 3 to 12, then 2% buffer B held from minute 12 to 15, followed by CH:FLOW_GRADIENT 2% buffer B to 85% buffer B from minute 15 to 16, then 85% buffer B held from CH:FLOW_GRADIENT minute 16-23 in order to re-equilibrate the column. CH:FLOW_RATE 400uL/minute CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE APCI MS:ION_MODE POSITIVE MS:MS_COMMENTS Selective reaction monitoring, SRM, of endogenous water-soluble metabolites was MS:MS_COMMENTS performed for steady state analysis. The positive ion mode ESI voltage was MS:MS_COMMENTS +4900. Dwell time was set to 3ms per SRM transition and 1.55 seconds was the MS:MS_COMMENTS total cycle time. Retention time 15-20 seconds. MultiQuant v2.1 software was MS:MS_COMMENTS used to in integrate peak areas for each metabolite SRM transition #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area intensity MS_METABOLITE_DATA_START Samples PTEN WT Rep1 PTEN WT Rep2 PTEN WT Rep3 PTEN KO Rep1 PTEN KO Rep2 PTEN KO Rep3 Factors Genotype:PTEN Wild-Type Genotype:PTEN Wild-Type Genotype:PTEN Wild-Type Genotype:PTEN knock-out Genotype:PTEN knock-out Genotype:PTEN knock-out Cystine 7574101.0 5871576.2 9106431.4 55636797.7 35079147.1 44897689.7 Cysteine 622319.1387 962551.1793 991136.9057 2945700.322 2717289.838 4647020.861 gamma-Glutamylcysteine 75264.3 420264.3 435933.9 1798001.1 1685253.0 2367334.3 Glutathione 105507.9019 93744.94363 115363.1545 419573.3506 521337.5937 415946.5429 Betaine 46784373.1 46393929.6 50596412.2 56766875.0 54313200.4 57996685.7 S-Adenosyl-L-Methionine 1494496.4 1530362.5 1825541.0 4170264.7 5793230.5 4515826.6 S-Adenosyl-L-Methioninamine 2795.2 9436.8 5714.4 22078.6 13115.6 28189.6 S-Adenosyl-L-Homocysteine 155240.8 184307.6 174184.5 234644.8 227756.2 281570.1 S-Ribosyl-L-Homocysteine 109855.1 110792.7 115031.2 251788.6 253937.6 263652.3 Homocysteine 66187.6 76531.7 67788.0 229993.6 195081.0 184255.4 7,8-Dihydrofolate 13418.9 11756.9 18195.7 48995.8 33160.5 38600.6 5-Methyl-THF 10599.0 11612.6 18979.8 25129.2 18955.9 26278.0 Choline 2742439.6 1969326.2 2033827.2 3998817.8 3651637.5 4204286.6 Phosphorylcholine 12983508.8 13310942.4 27768065.2 38527344.0 45708422.4 38661857.4 CDP-Choline 72768.2 72769.5 94053.1 194052.7 428598.7 436684.3 CDP-Ethanolamine 153655.5 88943.3 80867.7 169784.5 315389.5 307302.9 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PTEN WT Rep1 PTEN WT Rep2 PTEN WT Rep3 PTEN KO Rep1 PTEN KO Rep2 PTEN KO Rep3 Cystine 7574101.0 5871576.2 9106431.4 55636797.7 35079147.1 44897689.7 Cysteine 622319.1387 962551.1793 991136.9057 2945700.322 2717289.838 4647020.861 gamma-Glutamylcysteine 75264.3 420264.3 435933.9 1798001.1 1685253.0 2367334.3 Glutathione 105507.9019 93744.94363 115363.1545 419573.3506 521337.5937 415946.5429 Betaine 46784373.1 46393929.6 50596412.2 56766875.0 54313200.4 57996685.7 S-Adenosyl-L-Methionine 1494496.4 1530362.5 1825541.0 4170264.7 5793230.5 4515826.6 S-Adenosyl-L-Methioninamine 2795.2 9436.8 5714.4 22078.6 13115.6 28189.6 S-Adenosyl-L-Homocysteine 155240.8 184307.6 174184.5 234644.8 227756.2 281570.1 S-Ribosyl-L-Homocysteine 109855.1 110792.7 115031.2 251788.6 253937.6 263652.3 Homocysteine 66187.6 76531.7 67788.0 229993.6 195081.0 184255.4 7,8-Dihydrofolate 13418.9 11756.9 18195.7 48995.8 33160.5 38600.6 5-Methyl-THF 10599.0 11612.6 18979.8 25129.2 18955.9 26278.0 Choline 2742439.6 1969326.2 2033827.2 3998817.8 3651637.5 4204286.6 Phosphorylcholine 12983508.8 13310942.4 27768065.2 38527344.0 45708422.4 38661857.4 CDP-Choline 72768.2 72769.5 94053.1 194052.7 428598.7 436684.3 CDP-Ethanolamine 153655.5 88943.3 80867.7 169784.5 315389.5 307302.9 METABOLITES_END #END