Summary of study ST000058

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 PR000055. The data can be accessed directly via it's Project DOI: 10.21228/M83S3W This work is supported by NIH grant, U2C- DK119886.

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Study IDST000058
Study TitleMetabolite changes associated with methionine stress sensitivity of cancer (GC TOF MS analysis)
Study Typetimecourse study
Study SummaryThis West Coast Metabolomics Center pilot and feasibility project granted to Peter Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic dependence of cancer cells to explore development of novel unconventional therapeutic strategies that exploit dependence of cancer cells on methyl-donor abundance. The past few years have highlighted the role of altered metabolism in cancer. While mechanistic insight into changed metabolism in cancer is very limited, the importance of the metabolic pathway surrounding homocysteine and methionine for cancer cell proliferation has been known for over 30 years. These findings, generally summarized as methionine-dependence or methionine stress sensitivity, describe the phenomenon that most cancer cells cannot proliferate in growth medium where the amino acid methionine is replaced with its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells are unaffected by replacing methionine with homocysteine in the growth medium. For the past years we have been studying methionine dependence of breast and prostate cancer and demonstrated that methionine-dependence is caused by insufficient flux through this pathway to sustain synthesis of the downstream metabolite and the principal methyl-donor S-adenosylmethionine (SAM). We have isolated rare cell clones from MDA-MB468 breast cancer cells (referred to as MB468RES) that are no longer methionine dependent and proliferate in homocysteine medium. Interestingly, MB468RES have lost their ability for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES cell line pair confirms other observations showing that methionine dependence is tightly linked to tumorigenicity. Importantly, this cell line pair is an ideal model to identify metabolite signatures linked to cancer cell methionine dependence. We propose to characterize the metabolic changes triggered by the shift from normal growth medium to homocysteine medium in MB468 breast cancer cells and the methionine stress insensitive MB468RES derivatives. In addition we have developed cancer cell lines with inducible shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing synthesis of SAM from methionine and ATP. Inducible knockdown of MAT allows us to specifically reduce SAM synthesis. Our previous results suggest that SAM limitation is the critical trigger for cancer cell methionine dependence. Thus metabolite profiling using the MAT knockdown system will provide an independent dataset that together with metabolite profiles from the MB468 and MB468RES cell line pair will define critical metabolic profiles related to cancer cell methionine dependence.   In the current investigation, untargeted analysis of primary metabolites and complex lipids, coupled with quantitative analysis of methionine pathway intermediates (folate and respective derivatives, s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic flux will be conducted on MB468, MB468RES and MB468shRNA following the switch from methionine containing media to homocysteine containing media over the course of 0, 2, 4, 8, 12, 24 and 48 hours.   The primary objectives were to 1) characterize the metabolic response to methionine stress and SAM limitation and 2) correlate the metabolic signatures with cancer cell proliferation arrest and death. 
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
Address1315 Genome and Biomedical Sciences Facility 451 Health Sciences Drive Davis, CA 95616
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2014-06-11
Num Groups8
Total Subjects30
Study Comments2014-01-09 11:02:41.515
Raw Data AvailableYes
Raw Data File Type(s)binary files .peG,.cdf, .txt files
Uploaded File Size1.3 G
Analysis Type DetailGC-MS
Release Date2014-07-11
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M83S3W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000055
Project DOI:doi: 10.21228/M83S3W
Project Title:Metabolite changes associated with methionine stress sensitivity of cancer
Project Type:timecourse study
Project Summary:This West Coast Metabolomics Center pilot and feasibility project granted to Peter Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic dependence of cancer cells to explore development of novel unconventional therapeutic strategies that exploit dependence of cancer cells on methyl-donor abundance. The past few years have highlighted the role of altered metabolism in cancer. While mechanistic insight into changed metabolism in cancer is very limited, the importance of the metabolic pathway surrounding homocysteine and methionine for cancer cell proliferation has been known for over 30 years. These findings, generally summarized as methionine-dependence or methionine stress sensitivity, describe the phenomenon that most cancer cells cannot proliferate in growth medium where the amino acid methionine is replaced with its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells are unaffected by replacing methionine with homocysteine in the growth medium. For the past years we have been studying methionine dependence of breast and prostate cancer and demonstrated that methionine-dependence is caused by insufficient flux through this pathway to sustain synthesis of the downstream metabolite and the principal methyl-donor S-adenosylmethionine (SAM). We have isolated rare cell clones from MDA-MB468 breast cancer cells (referred to as MB468RES) that are no longer methionine dependent and proliferate in homocysteine medium. Interestingly, MB468RES have lost their ability for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES cell line pair confirms other observations showing that methionine dependence is tightly linked to tumorigenicity. Importantly, this cell line pair is an ideal model to identify metabolite signatures linked to cancer cell methionine dependence. We propose to characterize the metabolic changes triggered by the shift from normal growth medium to homocysteine medium in MB468 breast cancer cells and the methionine stress insensitive MB468RES derivatives. In addition we have developed cancer cell lines with inducible shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing synthesis of SAM from methionine and ATP. Inducible knockdown of MAT allows us to specifically reduce SAM synthesis. Our previous results suggest that SAM limitation is the critical trigger for cancer cell methionine dependence. Thus metabolite profiling using the MAT knockdown system will provide an independent dataset that together with metabolite profiles from the MB468 and MB468RES cell line pair will define critical metabolic profiles related to cancer cell methionine dependence. In the current investigation, untargeted analysis of primary metabolites and complex lipids, coupled with quantitative analysis of methionine pathway intermediates (folate and respective derivatives, s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic flux will be conducted on MB468, MB468RES and MB468shRNA following the switch from methionine containing media to homocysteine containing media over the course of 0, 2, 4, 8, 12, 24 and 48 hours. The primary objectives were to 1) characterize the metabolic response to methionine stress and SAM limitation and 2) correlate the metabolic signatures with cancer cell proliferation arrest and death.
Institute:University of California, Davis
Department:Genome and Biomedical Sciences Facility
Laboratory:WCMC Metabolomics Core
Last Name:Fiehn
First Name:Oliver
Address:1315 Genome and Biomedical Sciences Facility,451 Health Sciences Drive, Davis, CA 95616
Email:ofiehn@ucdavis.edu
Phone:(530) 754-8258
Funding Source:NIH U24DK097154

Subject:

Subject ID:SU000077
Subject Type:Human cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Subject type: Human cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA003046 140111dlvsa25_1Group 1: Methionine Treated - Group 1
SA003047 140111dlvsa20_1Group 1: Methionine Treated - Group 1
SA003048 140111dlvsa16_1Group 1: Methionine Treated - Group 1
SA003049 140111dlvsa14_1Group 1: Methionine Treated - Group 1
SA003050 140111dlvsa04_1Group 2: Homocysteine Treated-2 hours - Group 2
SA003051 140111dlvsa27_1Group 2: Homocysteine Treated-2 hours - Group 2
SA003052 140111dlvsa24_1Group 2: Homocysteine Treated-2 hours - Group 2
SA003053 140111dlvsa02_1Group 2: Homocysteine Treated-2 hours - Group 2
SA003054 140111dlvsa21_1Group 3: Homocysteine Treated- 4 hours - Group 3
SA003055 140111dlvsa15_1Group 3: Homocysteine Treated- 4 hours - Group 3
SA003056 140111dlvsa19_1Group 3: Homocysteine Treated- 4 hours - Group 3
SA003057 140111dlvsa30_1Group 3: Homocysteine Treated- 4 hours - Group 3
SA003058 140111dlvsa26_1Group 4: Homocysteine Treated- 8 hours - Group 4
SA003059 140111dlvsa05_1Group 4: Homocysteine Treated- 8 hours - Group 4
SA003060 140111dlvsa01_1Group 4: Homocysteine Treated- 8 hours - Group 4
SA003061 140111dlvsa06_1Group 4: Homocysteine Treated- 8 hours - Group 4
SA003062 140111dlvsa03_1Group 5: Homocysteine Treated- 12 hours - Group 5
SA003063 140111dlvsa12_1Group 5: Homocysteine Treated- 12 hours - Group 5
SA003064 140111dlvsa18_1Group 5: Homocysteine Treated- 12 hours - Group 5
SA003065 140111dlvsa17_1Group 5: Homocysteine Treated- 12 hours - Group 5
SA003066 140111dlvsa09_1Group 6: Homocysteine Treated- 24 hours - Group 6
SA003067 140111dlvsa28_1Group 6: Homocysteine Treated- 24 hours - Group 6
SA003068 140111dlvsa13_1Group 6: Homocysteine Treated- 24 hours - Group 6
SA003069 140111dlvsa29_1Group 6: Homocysteine Treated- 24 hours - Group 6
SA003070 140111dlvsa08_1Group 7: Homocysteine Treated- 48 hours - Group 7
SA003071 140111dlvsa11_1Group 7: Homocysteine Treated- 48 hours - Group 7
SA003072 140111dlvsa22_1Group 7: Homocysteine Treated- 48 hours - Group 7
SA003073 140111dlvsa23_1Group 7: Homocysteine Treated- 48 hours - Group 7
Showing results 1 to 28 of 28

Collection:

Collection ID:CO000060
Sample Type:Breast

Treatment:

Treatment ID:TR000078

Sample Preparation:

Sampleprep ID:SP000073
Sampleprep Protocol Filename:Protocol_for_primary_metabolites.docx
Sample Spiking:FAMEs

Combined analysis:

Analysis ID AN000096
Analysis type MS
Chromatography type GC
Chromatography system Agilent 6890N
Column Restek Corporation Rtx-5Sil MS
MS Type EI
MS instrument type GC-TOF
MS instrument name Leco GC-TOF
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH000064
Methods Filename:Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf
Instrument Name:Agilent 6890N
Column Name:Restek Corporation Rtx-5Sil MS
Column Pressure:7.7 PSI (initial condition)
Column Temperature:50 - 330C
Flow Rate:1 ml/min
Injection Temperature:50C ramped to 250C by 12C/s
Sample Injection:0.5l
Oven Temperature:50C for 1 min, then ramped at 20C/min to 330C, held constant for 5 min
Transferline Temperature:230C
Washing Buffer:Ethyl Acetate
Sample Loop Size:30 m length x 0.25 mm internal diameter
Randomization Order:Excel generated
Chromatography Type:GC

MS:

MS ID:MS000072
Analysis ID:AN000096
Instrument Name:Leco GC-TOF
Instrument Type:GC-TOF
MS Type:EI
Ion Mode:POSITIVE
Ion Source Temperature:250°C
Ionization Energy:70eV
Mass Accuracy:Nominal
Source Temperature:250°C
Scanning Range:80-500 Da
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