{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000077","ANALYSIS_ID":"AN000124","VERSION":"1","CREATED_ON":"2016-09-17"}, "PROJECT":{"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, 95616","EMAIL":"ofiehn@ucdavis.edu","PHONE":"(530) 754-8258","FUNDING_SOURCE":"NIH U24DK097154"}, "STUDY":{"STUDY_TITLE":"Metabolite changes associated with methionine stress sensitivity of cancer (CSH MS analysis)","STUDY_TYPE":"timecourse study","STUDY_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 Facility451 Health Sciences DriveDavis, CA","EMAIL":"ofiehn@ucdavis.edu","PHONE":"(530) 754-8258","NUM_GROUPS":"2","TOTAL_SUBJECTS":"71","STUDY_COMMENTS":"Lipidomics profiles for study"}, "SUBJECT":{"SUBJECT_TYPE":"breast cancer cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENOTYPE_STRAIN":"MDA-MB-468","SPECIES_GROUP":"Human"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_100uM Met _0 hours_1132014.d", "Factors":{"Treatment":"100uM Met","Timepoint":"0 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_100uM Met _0 hours_1132014.d", "Factors":{"Treatment":"100uM Met","Timepoint":"0 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_100uM Met _0 hours_1132014.d", "Factors":{"Treatment":"100uM Met","Timepoint":"0 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_100uM Met _0 hours_1132014.d", "Factors":{"Treatment":"100uM Met","Timepoint":"0 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _2 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"2 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _2 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"2 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _2 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"2 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_370uM Hcy _2 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"2 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _4 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"4 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _4 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"4 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _4 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"4 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_370uM Hcy _4 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"4 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _8 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"8 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _8 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"8 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _8 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"8 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _12 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"12 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _12 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"12 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _12 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"12 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_370uM Hcy _12 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"12 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _24 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"24 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _24 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"24 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _24 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"24 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_370uM Hcy _24 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"24 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_1-2_370uM Hcy _48 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"48 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_2-2_370uM Hcy _48 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"48 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_3-2_370uM Hcy _48 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"48 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_sample_4-2_370uM Hcy _48 hours_1132014.d", "Factors":{"Treatment":"370uM Hcy","Timepoint":"48 hours"} }, { "Subject ID":"-", "Sample ID":"CSH_BioRec_Plasma_01_1132014.d", "Factors":{"Treatment":"quality check","Timepoint":"quality check"} }, { "Subject ID":"-", "Sample ID":"CSH_BioRec_Plasma_02_1132014.d", "Factors":{"Treatment":"quality check","Timepoint":"quality check"} }, { "Subject ID":"-", "Sample ID":"CSH_BioRec_Plasma_03_1132014.d", "Factors":{"Treatment":"quality check","Timepoint":"quality check"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"-","COLLECTION_PROTOCOL_FILENAME":"Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics.pdf","SAMPLE_TYPE":"Breast Cancer Cell Culture Pellets"}, "TREATMENT":{"TREATMENT_SUMMARY":"-","TREATMENT_PROTOCOL_FILENAME":"Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics.pdf"}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"-","SAMPLEPREP_PROTOCOL_FILENAME":"Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics.pdf"}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"UPLC","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) Pre-Column","METHODS_FILENAME":"Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics.pdf","COLUMN_PRESSURE":"450-850 bar","COLUMN_TEMPERATURE":"65 C","FLOW_GRADIENT":"15% B to 99% B","FLOW_RATE":"0.6 mL/min","INTERNAL_STANDARD":"See data dictionary","RETENTION_TIME":"See data dictionary","SAMPLE_INJECTION":"1.67 uL","SOLVENT_A":"60:40 Acetonitrile:Water +10mM Ammonium Formate +10mM Formic Acid","SOLVENT_B":"9:1 Isopropanol:Acetonitrile +10mM Ammonium Formate +10mM Formic Acid","ANALYTICAL_TIME":"13 min","CAPILLARY_VOLTAGE":"3500","TIME_PROGRAM":"15 min","WEAK_WASH_SOLVENT_NAME":"Isopropanol","WEAK_WASH_VOLUME":"5 seconds","STRONG_WASH_SOLVENT_NAME":"Same","TARGET_SAMPLE_TEMPERATURE":"Autosampler temp 4 C","RANDOMIZATION_ORDER":"Excel"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"WCMC Metabolomics Core","SOFTWARE_VERSION":"MassHunter","DETECTOR_TYPE":"TOF MCP","DATA_FORMAT":".d"}, "MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","CAPILLARY_VOLTAGE":"3500","COLLISION_GAS":"Nitrogen","DRY_GAS_FLOW":"13 l/min","DRY_GAS_TEMP":"200","FRAGMENT_VOLTAGE":"175","FRAGMENTATION_METHOD":"Auto 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