Summary of Study ST003409
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 PR002111. The data can be accessed directly via it's Project DOI: 10.21228/M8RC0H This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003409 |
| Study Title | Impact of giredestrant on the lipid profile of MCF-7 breast cancer cells |
| Study Summary | Based on some recent data from a giredestrant resistance/sensitivity screen, we have an evolving hypothesis that exposure to giredestrant might be altering levels of polyunsaturated fatty acids, and perhaps otherwise altering lipid homeostasis in cells. The sensitivity to ferroptosis results from peroxidation of PUFAs-PLs. We thus investigated whether giredestrant altered PL homeostasis with a time course analysis of MCF-7 cell lipid profile. MCF-7 cells were treated with 0.6 nM of giredestrant and DMSO respectively on Day 0 and collected on Day 2, Day 7 and Day 14 for lipid profile analysis. Giredestrant appeared to provoke, in MCF-7 cells, a broad remodelling of lipids towards PUFA-containing species, specifically after longer duration (7 and 14 day) treatment. We observed that giredestrant increases PUFA-containing phospholipids at the expense of MUFA-phospholipids. |
| Institute | Genentech Inc. |
| Last Name | Wong |
| First Name | Weng Ruh |
| Address | 1 DNA Way, South San Francisco, CA 94080, USA |
| wongw24@gene.com | |
| Phone | 4089048962 |
| Submit Date | 2024-08-14 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-09-08 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002111 |
| Project DOI: | doi: 10.21228/M8RC0H |
| Project Title: | Inhibition of GPX4 enhances CDK4/6 inhibitor and endocrine therapy activity in breast cancer. |
| Project Type: | MS quantitative analysis |
| Project Summary: | CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. We performed two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulated sensitivity, with GPX4 the top sensitiser in both screens. Depletion or inhibition of GPX4 increased sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitised triple negative breast cancer models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation was promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer. |
| Institute: | Genentech Inc. |
| Last Name: | Wong |
| First Name: | Weng Ruh |
| Address: | 1 DNA Way, South San Francisco, CA 94080, USA |
| Email: | wongw24@gene.com |
| Phone: | 4089048962 |
| Contributors: | Herrera-Abrey MT, Guan J, Khalid U, Ning J, Costa MR, Chan J, Li Q, Fortin J-P, Perampalam P, Biton A, Sandoval W, Vijay J, Hafner M, Cutts R, Wilson G, Frankum J, Roumeliotis TI, Alexander J, Hickman O, Brough R, Haider S, Choudhary J, Lord CJ, Swain, A, Metcalfe C, Tuner NC |
Subject:
| Subject ID: | SU003535 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Genotype Strain: | MCF-7 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment | Time |
|---|---|---|---|---|
| SA376154 | T0_DMSO_2 | MCF7 Breast Cancer Cells | Control | Day_0 |
| SA376155 | T0_DMSO_1 | MCF7 Breast Cancer Cells | Control | Day_0 |
| SA376156 | T0_DMSO_4 | MCF7 Breast Cancer Cells | Control | Day_0 |
| SA376157 | T0_DMSO_3 | MCF7 Breast Cancer Cells | Control | Day_0 |
| SA376158 | T14_DMSO_4 | MCF7 Breast Cancer Cells | Control | Day_14 |
| SA376159 | T14_DMSO_3 | MCF7 Breast Cancer Cells | Control | Day_14 |
| SA376160 | T14_DMSO_2 | MCF7 Breast Cancer Cells | Control | Day_14 |
| SA376161 | T14_DMSO_1 | MCF7 Breast Cancer Cells | Control | Day_14 |
| SA376162 | T2_DMSO_2 | MCF7 Breast Cancer Cells | Control | Day_2 |
| SA376163 | T2_DMSO_3 | MCF7 Breast Cancer Cells | Control | Day_2 |
| SA376164 | T2_DMSO_4 | MCF7 Breast Cancer Cells | Control | Day_2 |
| SA376165 | T2_DMSO_1 | MCF7 Breast Cancer Cells | Control | Day_2 |
| SA376166 | T7_DMSO_4 | MCF7 Breast Cancer Cells | Control | Day_7 |
| SA376167 | T7_DMSO_3 | MCF7 Breast Cancer Cells | Control | Day_7 |
| SA376168 | T7_DMSO_2 | MCF7 Breast Cancer Cells | Control | Day_7 |
| SA376169 | T7_DMSO_1 | MCF7 Breast Cancer Cells | Control | Day_7 |
| SA376170 | T14_9545_1 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_14 |
| SA376171 | T14_9545_2 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_14 |
| SA376172 | T14_9545_3 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_14 |
| SA376173 | T14_9545_4 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_14 |
| SA376174 | T2_9545_4 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_2 |
| SA376175 | T2_9545_3 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_2 |
| SA376176 | T2_9545_2 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_2 |
| SA376177 | T2_9545_1 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_2 |
| SA376178 | T7_9545_2 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_7 |
| SA376179 | T7_9545_3 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_7 |
| SA376180 | T7_9545_4 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_7 |
| SA376181 | T7_9545_1 | MCF7 Breast Cancer Cells | Giredestrant_0.6nM | Day_7 |
| Showing results 1 to 28 of 28 |
Collection:
| Collection ID: | CO003528 |
| Collection Summary: | MCF7 were seeded at 4x106 cells in 17 T175 flasks in RPMI medium overnight. On day 0, the cells from 4 untreated flasks were trypsinized and washed; 2 x106 cells were collected and frozen |
| Collection Protocol Filename: | OBJ0042379_Giredestrant_protocol.pdf |
| Sample Type: | Breast cancer cells |
Treatment:
| Treatment ID: | TR003544 |
| Treatment Summary: | Time-points (n = 4; 2x10^6 cells for pelleting and submission) - Time 0 - DMSO @ 48h, 7 days, 14 days - GDC-9545 0.6 nM @ 48h, 7 days, 14 days - Total of 28 samples |
| Treatment Protocol Filename: | OBJ0042379_Giredestrant_protocol.pdf |
Sample Preparation:
| Sampleprep ID: | SP003542 |
| Sampleprep Summary: | Cells were homogenized in dichloromethane (DCM):methanol (1:1, v:v). After centrifuging, homogenate containing the same amount of proteins was transferred into a v-bottom glass tube. 0.5 ml water, 0.45 ml DCM and 1.0 ml methanol were added to the supernatant to form a single phase. After 30 minutes, isotope labeled internal standards were added to the mixture, followed by 0.45 ml DCM and 0.5 ml water. The mixture was centrifuged at 1000x g for 20 minutes. Phase separation was achieved after centrifuge. The bottom layer was then collected into a clean glass tube, and the upper layer was re-extracted by adding 1.8 ml of DCM. The bottom layer was combined and dried under a gentle stream of nitrogen. The residue was reconstituted in 300 μl of DCM:Methanol (1:1), 10 mM ammonium acetate for LipidyzerTM Platform direct infusion analysis [ref:Zhijun Cao, Thomas C. Schmitt, Vijayalakshmi Varma et al. Evaluation of the Performance of Lipidyzer Platform and Its Application in the Lipidomics Analysis in Mouse Heart and Liver; J. Proteome Res. 2020, 19, 7, 2742–2749] on AB Sciex 6500+ LC-MS/MS. Flow rate is set at 7 μl/min. The injection volume is 50 μl. The autosampler temperature was kept at 15 °C. Buffer A and B are the same as the reconstitution buffer [DCM:Methanol (1:1), 10 mM ammonium acetate]. Lipids concentrations were calculated by the LipidyzerTM platform based on the known concentrations of spiked internal standards. Heatmap was generated using R. |
Combined analysis:
| Analysis ID | AN005596 | AN005597 | AN005598 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | None (Direct infusion) | None (Direct infusion) | None (Direct infusion) |
| Chromatography system | Sciex QTRAP 6500+ with SelexION | Sciex QTRAP 6500+ with SelexION | Sciex QTRAP 6500+ |
| Column | none | none | none |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex 6500+ QTrap | ABI Sciex 6500+ QTrap | ABI Sciex 6500+ QTrap |
| Ion Mode | NEGATIVE | POSITIVE | POSITIVE |
| Units | nmol/300 ug protein | nmol/300 ug protein | nmol/300 ug protein |
Chromatography:
| Chromatography ID: | CH004253 |
| Chromatography Summary: | DMS on with switching pos/neg polarity |
| Chromatography Comments: | Reference: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15 |
| Instrument Name: | Sciex QTRAP 6500+ with SelexION |
| Column Name: | none |
| Column Temperature: | none |
| Flow Gradient: | none |
| Flow Rate: | none |
| Sample Injection: | 50 ul |
| Solvent A: | none |
| Solvent B: | none |
| Chromatography Type: | None (Direct infusion) |
| Chromatography ID: | CH004254 |
| Chromatography Summary: | DMS off with switching pos/neg polarity |
| Chromatography Comments: | Reference: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15 |
| Instrument Name: | Sciex QTRAP 6500+ |
| Column Name: | none |
| Column Temperature: | none |
| Flow Gradient: | none |
| Flow Rate: | none |
| Sample Injection: | 50 ul |
| Solvent A: | none |
| Solvent B: | none |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS005321 |
| Analysis ID: | AN005596 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This method is using a flow injection analysis (FIA): one injection with the SelexION voltages turned ON 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. The Negative ion mode detected the following lipid classes: LPE/LPC/PC/PE 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15 |
| Ion Mode: | NEGATIVE |
| MS ID: | MS005322 |
| Analysis ID: | AN005597 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This method is using a flow injection analysis (FIA): one injection with the SelexION voltages turned ON 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. The Positive ion mode detected the following lipid classes: SM 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids. Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15 |
| Ion Mode: | POSITIVE |
| MS ID: | MS005323 |
| Analysis ID: | AN005598 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | DI-MS/MS Analysis 1. A QTRAP® system with SelexION Technology (SCIEX) is used for targeted profiling (SCIEX, MA, USA). 2. This second method is also using a flow injection analysis (FIA): a separate injection with the SelexION voltages turned OFF. 3. The lipid molecular species are measured using multiple reaction monitoring (MRM) and positive/negative switching. Positive ion mode detected the following lipid classes: DAG/CE/CER/LCER/HCER/TAG. Negative ion mode detected the following lipid classes: FFA (data not used in this study) 4. A flow injection analysis (FIA) setup is employed by using the LC to flow at an isocratic rate of 7 μL/min with a ramp up to 30 μL/min for the last 2 min of the experiment to allow for washing. 5. Data acquisition is around 20 min per sample, and 50 μL of the reconstituted sample is infused and the area under the flat infusion line reported and corrected to the appropriate internal standard . 6. Samples are quantified using the LWM software which reports all the detected lipids. Refence: Ubhi, B.K. (2018). Direct Infusion-Tandem Mass Spectrometry (DI-MS/MS) Analysis of Complex Lipids in Human Plasma and Serum Using the Lipidyzer™ Platform. In: Giera, M. (eds) Clinical Metabolomics. Methods in Molecular Biology, vol 1730. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7592-1_15 |
| Ion Mode: | POSITIVE |
