Summary of Study ST003410
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 | ST003410 |
| Study Title | Lipidomics Analysis of ER+ Breast Cancer Cells Treated with Giredestrant and Palbociclib |
| Study Summary | We observed in a previous experiment that giredestrant treatment had a profound impact on the lipid profile of MCF-7 cells, in particular through elevating PUFAs at the apparent expense of MUFAs. We hypothesize that this shift in PUFA/MUFA ratio underlies giredestrant-induced sensitivity to GPX4 inhibition. Emerging data from our lab and other suggests that palbociclib, a key combination partner for giredestrant in the clinic, also sensitizes to GPX4 inhibition, and that the combination of giredestrant and palbo may synergize to creating an even higher sensitivity to GPX4i. Here, we aim to directly compare the effects on the lipidome of giredestrant and palbociclib, in MCF-7 cells (the discovery cell line where we already have some lipidomics data), and also T-47D cells, in which giredestrant, palbociclib and their combination drive a very profound sensitization to GPX4i, that exceeds what was observed in MCF-7 cells. The cells were treated with DMSO, 1 nM giredestrant, 200 nM palbociclib, or 1nM giredestrant plus 200nM palbociclib in quadruplicates, and collected on Day 7 for lipid analysis. Despite their differences under basal conditions, drug treatments altered the lipid profiles of MCF7 and T47D cells in a similar manner, with 200nM palbociclib at the 7 day time point having a less pronounced effect than giredestrant, and with the greatest lipid changes occurring with the combination of palbociclib plus giredestrant. PUFA-ePLs were likewise elevated by giredestrant and/or palbociclib in both cell lines, with the combined action of both drugs generally driving greatest increases in individual PUFA-linked ePL species. Palbociclib combination with giredestrant enhance the accumulation of PUFAs-phospholipids compared to the single treatments. |
| 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 | 2024-08-15 |
| 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: | SU003536 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Genotype Strain: | MCF-7 and T-47D |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment | Time |
|---|---|---|---|---|
| SA376182 | MCF7_DMSO_2 | MCF7 breast cancer cells | Control | Day_7 |
| SA376183 | MCF7_DMSO_4 | MCF7 breast cancer cells | Control | Day_7 |
| SA376184 | MCF7_DMSO_1 | MCF7 breast cancer cells | Control | Day_7 |
| SA376185 | MCF7_DMSO_3 | MCF7 breast cancer cells | Control | Day_7 |
| SA376190 | MCF7_9plusP_4 | MCF7 breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376191 | MCF7_9plusP_1 | MCF7 breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376192 | MCF7_9plusP_2 | MCF7 breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376193 | MCF7_9plusP_3 | MCF7 breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376186 | MCF7_9545_1 | MCF7 breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376187 | MCF7_9545_2 | MCF7 breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376188 | MCF7_9545_4 | MCF7 breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376189 | MCF7_9545_3 | MCF7 breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376194 | MCF7_Palbo_4 | MCF7 breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376195 | MCF7_Palbo_1 | MCF7 breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376196 | MCF7_Palbo_3 | MCF7 breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376197 | MCF7_Palbo_2 | MCF7 breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376198 | T47D_DMSO_1 | T47D breast cancer cells | Control | Day_7 |
| SA376199 | T47D_DMSO_4 | T47D breast cancer cells | Control | Day_7 |
| SA376200 | T47D_DMSO_2 | T47D breast cancer cells | Control | Day_7 |
| SA376201 | T47D_DMSO_3 | T47D breast cancer cells | Control | Day_7 |
| SA376206 | T47D_9plusP_3 | T47D breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376207 | T47D_9plusP_1 | T47D breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376208 | T47D_9plusP_4 | T47D breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376209 | T47D_9plusP_2 | T47D breast cancer cells | Giredestrant_1nM_plus_Palbociclib_200nM | Day_7 |
| SA376202 | T47D_9545_3 | T47D breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376203 | T47D_9545_2 | T47D breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376204 | T47D_9545_1 | T47D breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376205 | T47D_9545_4 | T47D breast cancer cells | Giredestrant_1nM | Day_7 |
| SA376210 | T47D_Palbo_3 | T47D breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376211 | T47D_Palbo_2 | T47D breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376212 | T47D_Palbo_1 | T47D breast cancer cells | Palbociclib_200nM | Day_7 |
| SA376213 | T47D_Palbo_4 | T47D breast cancer cells | Palbociclib_200nM | Day_7 |
| Showing results 1 to 32 of 32 |
Collection:
| Collection ID: | CO003529 |
| Collection Summary: | All cell lines were obtained from American Type Culture Collection (ATCC) or Asterand and maintained according to the supplier's instructions. MCF-7 and T47D cells were seeded at same density, 4x106 cells in RPMI medium in T175 flask (16 flasks each for each cell line) overnight. On day 0, the cells were treated with DMSO, 1 nM giredestrant, 200 nM palbociclib, or 1nM giredestrant plus 200nM palbociclib in quadruplicates. On day 4, after the cells were passaged, and 4x106 cells from each flask were reseeded into another T175, and continued with the same treatment for 3 more days. On day 7, the cells were trypsinized and counted, and 1.5-2 x106 cells from each flask were collected and frozen for lipid analysis. |
| Sample Type: | Breast cancer cells |
Treatment:
| Treatment ID: | TR003545 |
| Treatment Summary: | Time-points (n = 4; 2x10^6 cells for pelleting and submission) - Time: 7 days - Treatment: DMSO; 1 nM Giredestrant (GDC-9545); 200nM Palbociclib; Giredestrant (GDC-9545) + Palbo Total samples: 32 |
Sample Preparation:
| Sampleprep ID: | SP003543 |
| 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 | AN005599 | AN005600 | AN005601 |
|---|---|---|---|
| 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/250 ug protein | nmol/250 ug protein | nmol/250 ug protein |
Chromatography:
| Chromatography ID: | CH004255 |
| 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: | CH004256 |
| 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: | MS005324 |
| Analysis ID: | AN005599 |
| 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: | MS005325 |
| Analysis ID: | AN005600 |
| 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: | MS005326 |
| Analysis ID: | AN005601 |
| 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 |
