Summary of Study ST002852

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 PR001786. The data can be accessed directly via it's Project DOI: 10.21228/M8VB1G 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	ST002852
Study Title	MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer
Study Summary	Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effect of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides new insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.
Institute	University of California, Los Angeles
Department	Biological Chemistry
Laboratory	Heather Christofk
Last Name	Matulionis
First Name	Nedas
Address	615 Charles E Young Dr S, BSRB 354-05
Email	nmatulionis@mednet.ucla.edu
Phone	3102060163
Submit Date	2023-09-07
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-09-11
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001786
Project DOI:	doi: 10.21228/M8VB1G
Project Title:	MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer
Project Summary:	Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effect of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides new insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.
Institute:	University of California, Los Angeles
Department:	Biological Chemistry
Laboratory:	Heather Christofk
Last Name:	Matulionis
First Name:	Nedas
Address:	615 Charles E Young Dr S, BSRB 354-05
Email:	nmatulionis@mednet.ucla.edu
Phone:	3102060163

Subject:

Subject ID:	SU002964
Subject Type:	Mammal
Subject Species:	Mus musculus
Genotype Strain:	NSG

Factors:

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

mb_sample_id	local_sample_id	Treatment	Technical Replicate (Region of Tumor)
SA308642	tumor-enzalutamide-3-01	Enzalutamide	1
SA308643	tumor-enzalutamide-4-01	Enzalutamide	1
SA308644	tumor-enzalutamide-5-01	Enzalutamide	1
SA308645	tumor-enzalutamide-1-01	Enzalutamide	1
SA308646	tumor-enzalutamide-2-01	Enzalutamide	1
SA308647	tumor-enzalutamide-4-02	Enzalutamide	2
SA308648	tumor-enzalutamide-5-02	Enzalutamide	2
SA308649	tumor-enzalutamide-2-02	Enzalutamide	2
SA308650	tumor-enzalutamide-3-02	Enzalutamide	2
SA308651	tumor-enzalutamide-1-02	Enzalutamide	2
SA308652	tumor-veh-2-01	Vehicle	1
SA308653	tumor-veh-3-01	Vehicle	1
SA308654	tumor-veh-1-01	Vehicle	1
SA308655	tumor-veh-1-02	Vehicle	2
SA308656	tumor-veh-3-02	Vehicle	2
SA308657	tumor-veh-2-02	Vehicle	2

Showing results 1 to 16 of 16

Showing results 1 to 16 of 16

Collection:

Collection ID:	CO002957
Collection Summary:	Tumors were collected after 10 days of treatment and prepared for histology, protein extraction, and metabolite extraction.
Sample Type:	Prostate

Treatment:

Treatment ID:	TR002973
Treatment Summary:	7 million 16D cells were implanted subcutaneously with 100 μl Matrigel (Corning) into NSG mice to form primary tumors. Primary tumors were harvested, minced, and re-implanted (20 - 80 mg of minced tumor tissue with 100 μl Matrigel per mouse) into NSG mice. 16D tumor-bearing mice were treated by oral gavage with 10 mg/kg/day of Enzalutamide in the vehicle (1% carboxymethyl cellulose, 0.5% Tween 80, and 5% dimethylsulfoxide) or the vehicle only, with a two-days-on/one-day-off schedule.

Treatment ID:

TR002973

Treatment Summary:

7 million 16D cells were implanted subcutaneously with 100 μl Matrigel (Corning) into NSG mice to form primary tumors. Primary tumors were harvested, minced, and re-implanted (20 - 80 mg of minced tumor tissue with 100 μl Matrigel per mouse) into NSG mice. 16D tumor-bearing mice were treated by oral gavage with 10 mg/kg/day of Enzalutamide in the vehicle (1% carboxymethyl cellulose, 0.5% Tween 80, and 5% dimethylsulfoxide) or the vehicle only, with a two-days-on/one-day-off schedule.

Sample Preparation:

Sampleprep ID:	SP002970
Sampleprep Summary:	After tumor dissection, a maximum of 30mg of tissue was weighed, snap frozen, and stored at -80C until metabolite extraction. To extract metabolites, weighed tumor tissue was added to a bead tube (Thermo Fisher Scientific) containing 1ml 80% methanol plus 10mM potassium trifluoromethanesulfonate (TMSO) internal standard on ice. Samples were homogenized for 1 minute at max speed on a bead homogenizer (Thermo Fisher Scientific). Bead tubes were spun at 17000g at 4C for 10 minutes. The supernatant was transferred to an Eppendorf tube and spun at 17000g at 4C for 10 minutes. A volume of extraction equivalent to 3mg of tumor tissue was transferred to an ABC vial (Thermo Fisher Scientific). All volumes were normalized to 500 µl with 80% methanol containing TMSO internal standard. 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 minutes at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis.

Sampleprep ID:

SP002970

Sampleprep Summary:

After tumor dissection, a maximum of 30mg of tissue was weighed, snap frozen, and stored at -80C until metabolite extraction. To extract metabolites, weighed tumor tissue was added to a bead tube (Thermo Fisher Scientific) containing 1ml 80% methanol plus 10mM potassium trifluoromethanesulfonate (TMSO) internal standard on ice. Samples were homogenized for 1 minute at max speed on a bead homogenizer (Thermo Fisher Scientific). Bead tubes were spun at 17000g at 4C for 10 minutes. The supernatant was transferred to an Eppendorf tube and spun at 17000g at 4C for 10 minutes. A volume of extraction equivalent to 3mg of tumor tissue was transferred to an ABC vial (Thermo Fisher Scientific). All volumes were normalized to 500 µl with 80% methanol containing TMSO internal standard. 80% MeOH was evaporated from the ABC vials using the EZ-2Elite evaporator (Genevac) and samples were stored at -80°C until analysis. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile (ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 minutes at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials. 10 µL of these metabolite solutions were injected per analysis.

Combined analysis:

Analysis ID	AN004672	AN004673
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Peak Area	Peak Area

Chromatography:

Chromatography ID:	CH003516
Chromatography Summary:	Samples were run on a Vanquish (Thermo Fisher Scientific) UHPLC system with mobile phase A (20mM ammonium carbonate, pH 9.7) and mobile phase B (100% ACN) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 minutes followed by a linear gradient from 80% A to 20% A from 20 minutes to 20.5 minutes. 20% A was then held from 20.5 minutes to 28 minutes.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm,5um)
Column Temperature:	35°C
Flow Gradient:	Linear gradient was as follows: 20%A to 80%A (0-20 min), 80%A to 20%A (20-20.5 min), hold 20%A (20.5-28.0 min).
Flow Rate:	150 µL/min
Solvent A:	20 mM Ammonium carbonate, pH 9.7
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004419
Analysis ID:	AN004672
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Fisher Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard12. These “.mzXML” files were imported into the MZmine 2 software package13. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module14 and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor15 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	POSITIVE

MS ID:	MS004420
Analysis ID:	AN004673
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The UHPLC was coupled to a Q-Exactive (Thermo Fisher Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard12. These “.mzXML” files were imported into the MZmine 2 software package13. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module14 and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor15 to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well.
Ion Mode:	NEGATIVE