{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000058","ANALYSIS_ID":"AN000096","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 Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic of cancer cells to explore development of novel unconventional therapeutic that exploit dependence of cancer cells on methyl-donor abundance. The past few have highlighted the role of altered metabolism in cancer. While mechanistic into changed metabolism in cancer is very limited, the importance of the pathway surrounding homocysteine and methionine for cancer cell proliferation been known for over 30 years. These findings, generally summarized as or methionine stress sensitivity, describe the phenomenon that most cancer cannot proliferate in growth medium where the amino acid methionine is replaced its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells unaffected by replacing methionine with homocysteine in the growth medium. For past years we have been studying methionine dependence of breast and prostate and demonstrated that methionine-dependence is caused by insufficient flux this pathway to sustain synthesis of the downstream metabolite and the methyl-donor S-adenosylmethionine (SAM). We have isolated rare cell clones from breast cancer cells (referred to as MB468RES) that are no longer methionine and proliferate in homocysteine medium. Interestingly, MB468RES have lost their for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES line pair confirms other observations showing that methionine dependence is linked to tumorigenicity. Importantly, this cell line pair is an ideal model to metabolite signatures linked to cancer cell methionine dependence. We propose characterize the metabolic changes triggered by the shift from normal growth to homocysteine medium in MB468 breast cancer cells and the methionine stress MB468RES derivatives. In addition we have developed cancer cell lines with shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing of SAM from methionine and ATP. Inducible knockdown of MAT allows us to reduce SAM synthesis. Our previous results suggest that SAM limitation is the trigger for cancer cell methionine dependence. Thus metabolite profiling using MAT knockdown system will provide an independent dataset that together with profiles from the MB468 and MB468RES cell line pair will define critical profiles related to cancer cell methionine dependence. In the current untargeted analysis of primary metabolites and complex lipids, coupled with analysis of methionine pathway intermediates (folate and respective s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic will be conducted on MB468, MB468RES and MB468shRNA following the switch from containing media to homocysteine containing media over the course of 0, 2, 4, 12, 24 and 48 hours. The primary objectives were to 1) characterize the response to methionine stress and SAM limitation and 2) correlate the metabolic 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 (GC MS analysis)","STUDY_TYPE":"timecourse study","STUDY_SUMMARY":"This West Coast Metabolomics Center pilot and feasibility project granted to Kaiser (UC Irvine), aims to achieve understanding of a unique metabolic of cancer cells to explore development of novel unconventional therapeutic that exploit dependence of cancer cells on methyl-donor abundance. The past few have highlighted the role of altered metabolism in cancer. While mechanistic into changed metabolism in cancer is very limited, the importance of the pathway surrounding homocysteine and methionine for cancer cell proliferation been known for over 30 years.These findings, generally summarized as or methionine stress sensitivity, describe the phenomenon that most cancer cannot proliferate in growth medium where the amino acid methionine is replaced its direct metabolic precursor homocysteine. Importantly, non-tumorigenic cells unaffected by replacing methionine with homocysteine in the growth medium. For past years we have been studying methionine dependence of breast and prostate and demonstrated that methionine-dependence is caused by insufficient flux this pathway to sustain synthesis of the downstream metabolite and the methyl-donor S-adenosylmethionine (SAM).We have isolated rare cell clones from breast cancer cells (referred to as MB468RES) that are no longer methionine and proliferate in homocysteine medium. Interestingly, MB468RES have lost their for anchorage independent growth, a hallmark of cancer. The MB468 and MB468RES line pair confirms other observations showing that methionine dependence is linked to tumorigenicity. Importantly, this cell line pair is an ideal model to metabolite signatures linked to cancer cell methionine dependence. We propose characterize the metabolic changes triggered by the shift from normal growth to homocysteine medium in MB468 breast cancer cells and the methionine stress MB468RES derivatives. In addition we have developed cancer cell lines with shRNAs targeting methionine adenosyltransferase (MAT), the enzyme catalyzing of SAM from methionine and ATP. Inducible knockdown of MAT allows us to reduce SAM synthesis. Our previous results suggest that SAM limitation is the trigger for cancer cell methionine dependence. Thus metabolite profiling using MAT knockdown system will provide an independent dataset that together with profiles from the MB468 and MB468RES cell line pair will define critical profiles related to cancer cell methionine dependence. In the current untargeted analysis of primary metabolites and complex lipids, coupled with analysis of methionine pathway intermediates (folate and respective s-adenosylmethoinine, s-adenosylhomocysteine, choline, betaine) and metabolic will be conducted on MB468, MB468RES and MB468shRNA following the switch from containing media to homocysteine containing media over the course of 0, 2, 4, 12, 24 and 48 hours. The primary objectives were to 1) characterize the response to methionine stress and SAM limitation and 2) correlate the metabolic 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":"8","TOTAL_SUBJECTS":"30","STUDY_COMMENTS":"2014-01-09 11:02:41.515"},

"SUBJECT":{"SUBJECT_TYPE":"breast cancer cells-MDA-MB-468","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","SPECIES_GROUP":"Human"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _2 hours",
"Sample ID":"140111dlvsa02_1",
"Factors":{"Treatment":"Group 2; Homocysteine Treated-2 hours - Group 2"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _2 hours",
"Sample ID":"140111dlvsa04_1",
"Factors":{"Treatment":"Group 2; Homocysteine Treated-2 hours - Group 2"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _2 hours",
"Sample ID":"140111dlvsa24_1",
"Factors":{"Treatment":"Group 2; Homocysteine Treated-2 hours - Group 2"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _2 hours",
"Sample ID":"140111dlvsa27_1",
"Factors":{"Treatment":"Group 2; Homocysteine Treated-2 hours - Group 2"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _4 hours",
"Sample ID":"140111dlvsa15_1",
"Factors":{"Treatment":"Group 3; Homocysteine Treated- 4 hours - Group 3"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _4 hours",
"Sample ID":"140111dlvsa19_1",
"Factors":{"Treatment":"Group 3; Homocysteine Treated- 4 hours - Group 3"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _4 hours",
"Sample ID":"140111dlvsa21_1",
"Factors":{"Treatment":"Group 3; Homocysteine Treated- 4 hours - Group 3"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _4 hours",
"Sample ID":"140111dlvsa30_1",
"Factors":{"Treatment":"Group 3; Homocysteine Treated- 4 hours - Group 3"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _8 hours",
"Sample ID":"140111dlvsa01_1",
"Factors":{"Treatment":"Group 4; Homocysteine Treated- 8 hours - Group 4"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _8 hours",
"Sample ID":"140111dlvsa05_1",
"Factors":{"Treatment":"Group 4; Homocysteine Treated- 8 hours - Group 4"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _8 hours",
"Sample ID":"140111dlvsa06_1",
"Factors":{"Treatment":"Group 4; Homocysteine Treated- 8 hours - Group 4"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _8 hours",
"Sample ID":"140111dlvsa26_1",
"Factors":{"Treatment":"Group 4; Homocysteine Treated- 8 hours - Group 4"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _12 hours",
"Sample ID":"140111dlvsa03_1",
"Factors":{"Treatment":"Group 5; Homocysteine Treated- 12 hours - Group 5"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _12 hours",
"Sample ID":"140111dlvsa12_1",
"Factors":{"Treatment":"Group 5; Homocysteine Treated- 12 hours - Group 5"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _12 hours",
"Sample ID":"140111dlvsa17_1",
"Factors":{"Treatment":"Group 5; Homocysteine Treated- 12 hours - Group 5"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _12 hours",
"Sample ID":"140111dlvsa18_1",
"Factors":{"Treatment":"Group 5; Homocysteine Treated- 12 hours - Group 5"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _24 hours",
"Sample ID":"140111dlvsa09_1",
"Factors":{"Treatment":"Group 6; Homocysteine Treated- 24 hours - Group 6"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _24 hours",
"Sample ID":"140111dlvsa13_1",
"Factors":{"Treatment":"Group 6; Homocysteine Treated- 24 hours - Group 6"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _24 hours",
"Sample ID":"140111dlvsa28_1",
"Factors":{"Treatment":"Group 6; Homocysteine Treated- 24 hours - Group 6"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _24 hours",
"Sample ID":"140111dlvsa29_1",
"Factors":{"Treatment":"Group 6; Homocysteine Treated- 24 hours - Group 6"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_370uM homocysteine _48 hours",
"Sample ID":"140111dlvsa08_1",
"Factors":{"Treatment":"Group 7; Homocysteine Treated- 48 hours - Group 7"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_370uM homocysteine _48 hours",
"Sample ID":"140111dlvsa11_1",
"Factors":{"Treatment":"Group 7; Homocysteine Treated- 48 hours - Group 7"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_370uM homocysteine _48 hours",
"Sample ID":"140111dlvsa22_1",
"Factors":{"Treatment":"Group 7; Homocysteine Treated- 48 hours - Group 7"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_370uM homocysteine _48 hours",
"Sample ID":"140111dlvsa23_1",
"Factors":{"Treatment":"Group 7; Homocysteine Treated- 48 hours - Group 7"}
},
{
"Subject ID":"MDA-MB-468_sample_2-1_100uM methionine _0 hours",
"Sample ID":"140111dlvsa14_1",
"Factors":{"Treatment":"Group 1; Methionine Treated - Group 1"}
},
{
"Subject ID":"MDA-MB-468_sample_1-1_100uM methionine _0 hours",
"Sample ID":"140111dlvsa16_1",
"Factors":{"Treatment":"Group 1; Methionine Treated - Group 1"}
},
{
"Subject ID":"MDA-MB-468_sample_4-1_100uM methionine _0 hours",
"Sample ID":"140111dlvsa20_1",
"Factors":{"Treatment":"Group 1; Methionine Treated - Group 1"}
},
{
"Subject ID":"MDA-MB-468_sample_3-1_100uM methionine _0 hours",
"Sample ID":"140111dlvsa25_1",
"Factors":{"Treatment":"Group 1; Methionine Treated - Group 1"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"-"},

"TREATMENT":{"TREATMENT_SUMMARY":"-"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"-","SAMPLEPREP_PROTOCOL_FILENAME":"Protocol_for_primary_metabolites.docx","SAMPLE_SPIKING":"FAMEs"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 6890N","COLUMN_NAME":"Restek Corporation Rtx-5Sil MS","METHODS_FILENAME":"Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf","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"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"WCMC Metabolomics Core","ACQUISITION_PARAMETERS_FILE":"Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf","SOFTWARE_VERSION":"ChromaTOF","OPERATOR_NAME":"Luis Valdiviez","PROCESSING_PARAMETERS_FILE":"Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf","DETECTOR_TYPE":"TOF","DATA_FORMAT":".peg, .txt, .cdf"},

"MS":{"MS_COMMENTS":"-","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"},

"MS_METABOLITE_DATA":{
"Units":"Peak area",

"Data":[{"Metabolite":"1-hexadecanol","140111dlvsa14_1":"186.0000","140111dlvsa16_1":"178.0000","140111dlvsa20_1":"87.0000","140111dlvsa25_1":"277.0000","140111dlvsa02_1":"304.0000","140111dlvsa04_1":"192.0000","140111dlvsa24_1":"233.0000","140111dlvsa27_1":"149.0000","140111dlvsa15_1":"146.0000","140111dlvsa19_1":"202.0000","140111dlvsa21_1":"198.0000","140111dlvsa30_1":"211.0000","140111dlvsa01_1":"242.0000","140111dlvsa05_1":"219.0000","140111dlvsa06_1":"206.0000","140111dlvsa26_1":"75.0000","140111dlvsa03_1":"113.0000","140111dlvsa12_1":"185.0000","140111dlvsa17_1":"193.0000","140111dlvsa18_1":"201.0000","140111dlvsa09_1":"61.0000","140111dlvsa13_1":"74.0000","140111dlvsa28_1":"246.0000","140111dlvsa29_1":"250.0000","140111dlvsa08_1":"237.0000","140111dlvsa11_1":"169.0000","140111dlvsa22_1":"145.0000","140111dlvsa23_1":"270.0000"},{"Metabolite":"1-monoolein","140111dlvsa14_1":"672.0000","140111dlvsa16_1":"956.0000","140111dlvsa20_1":"880.0000","140111dlvsa25_1":"654.0000","140111dlvsa02_1":"919.0000","140111dlvsa04_1":"760.0000","140111dlvsa24_1":"854.0000","140111dlvsa27_1":"818.0000","140111dlvsa15_1":"1343.0000","140111dlvsa19_1":"722.0000","140111dlvsa21_1":"787.0000","140111dlvsa30_1":"1210.0000","140111dlvsa01_1":"867.0000","140111dlvsa05_1":"966.0000","140111dlvsa06_1":"1203.0000","140111dlvsa26_1":"1480.0000","140111dlvsa03_1":"924.0000","140111dlvsa12_1":"1567.0000","140111dlvsa17_1":"1904.0000","140111dlvsa18_1":"1003.0000","140111dlvsa09_1":"1086.0000","140111dlvsa13_1":"1046.0000","140111dlvsa28_1":"1423.0000","140111dlvsa29_1":"946.0000","140111dlvsa08_1":"1114.0000","140111dlvsa11_1":"1679.0000","140111dlvsa22_1":"2553.0000","140111dlvsa23_1":"2656.0000"},{"Metabolite":"2-aminoadipic acid","140111dlvsa14_1":"286.0000","140111dlvsa16_1":"316.0000","140111dlvsa20_1":"222.0000","140111dlvsa25_1":"301.0000","140111dlvsa02_1":"386.0000","140111dlvsa04_1":"290.0000","140111dlvsa24_1":"132.0000","140111dlvsa27_1":"180.0000","140111dlvsa15_1":"134.0000","140111dlvsa19_1":"312.0000","140111dlvsa21_1":"211.0000","140111dlvsa30_1":"128.0000","140111dlvsa01_1":"340.0000","140111dlvsa05_1":"130.0000","140111dlvsa06_1":"233.0000","140111dlvsa26_1":"215.0000","140111dlvsa03_1":"269.0000","140111dlvsa12_1":"270.0000","140111dlvsa17_1":"160.0000","140111dlvsa18_1":"230.0000","140111dlvsa09_1":"263.0000","140111dlvsa13_1":"135.0000","140111dlvsa28_1":"157.0000","140111dlvsa29_1":"135.0000","140111dlvsa08_1":"190.0000","140111dlvsa11_1":"165.0000","140111dlvsa22_1":"155.0000","140111dlvsa23_1":"205.0000"},{"Metabolite":"2-deoxyerythritol","140111dlvsa14_1":"1333.0000","140111dlvsa16_1":"1446.0000","140111dlvsa20_1":"3120.0000","140111dlvsa25_1":"2368.0000","140111dlvsa02_1":"1404.0000","140111dlvsa04_1":"2292.0000","140111dlvsa24_1":"2372.0000","140111dlvsa27_1":"2380.0000","140111dlvsa15_1":"1677.0000","140111dlvsa19_1":"2761.0000","140111dlvsa21_1":"2831.0000","140111dlvsa30_1":"2537.0000","140111dlvsa01_1":"1806.0000","140111dlvsa05_1":"1397.0000","140111dlvsa06_1":"2371.0000","140111dlvsa26_1":"2496.0000","140111dlvsa03_1":"2502.0000","140111dlvsa12_1":"2699.0000","140111dlvsa17_1":"2326.0000","140111dlvsa18_1":"1347.0000","140111dlvsa09_1":"2541.0000","140111dlvsa13_1":"1211.0000","140111dlvsa28_1":"1507.0000","140111dlvsa29_1":"2451.0000","140111dlvsa08_1":"1314.0000","140111dlvsa11_1":"1103.0000","140111dlvsa22_1":"2925.0000","140111dlvsa23_1":"1214.0000"},{"Metabolite":"2-hydroxyglutaric 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