{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000644","ANALYSIS_ID":"AN000976","VERSION":"1","CREATED_ON":"June 25, 2017, 10:21 pm"},

"PROJECT":{"PROJECT_TITLE":"Role of the Serine Biosynthesis Pathway in Supporting the Warburg Effect of Pancreatic Cancer Cells","PROJECT_SUMMARY":"Pancreatic cancer cells metabolize glucose differently than normal adult cells, relying on aerobic glycolysis even in oxygen-rich environments. This phenomenon, known as the Warburg effect, is the basis of PET scans for tumor imaging and diagnosis, but a definitive explanation for how this benefits cancer cells has remained elusive, and altered cell metabolism has not been fully exploited for therapeutic benefit. The Warburg effect is accompanied by expression of the M2 isoform of pyruvate kinase (PK); while many differentiated normal cells express PKM1, proliferating cells, including all cancer cells, express PKM1. We have generated both normal cell lines and pancreatic cancer cell lines that can be genetically controlled to express either PKM1 or PKM2. While normal proliferating cells stop proliferating when forced to express PKM1 rather than PKM2, pancreatic cancer cells proliferate just as rapidly with forced PKM1 expression. Preliminary data shows that pancreatic cancer cells upregulate the serine biosynthesis pathway during forced PKM1 expression. To probe the role of the serine biosynthesis pathway in supporting cancer proliferation in the context of isoform-specific PK expression, we have targeted genes in the serine biosynthesis pathway using the CRISPR/Cas9 system and generated pancreatic cancer knockout cell lines. The proposed research will use isotope-labeled precursors and genetic engineering to identify the metabolic dependencies of pancreatic cancer cells. Genetically engineered pancreatic cancer cell lines cultured with 13C-glucose, 13C-glutamine, or 13C-serine will be extracted and sent to the Mayo Clinic Metabolomics Resource Core for isotopic enrichment analysis of various amino acids, TCA cycle metabolites, fatty acids, and sphingolipids. This work will provide crucial first insight for altered metabolism of pancreatic cancer cells that can lead to novel metabolic targets for effectively treating pancreatic cancer.","INSTITUTE":"Mayo Clinic","LAST_NAME":"Lunt","FIRST_NAME":"Sophia","ADDRESS":"Michigan State University 410B Biochemistry Building 603 Wilson Road East Lansing, MI 48824","EMAIL":"sophia@msu.edu","PHONE":"517-432-4886"},

"STUDY":{"STUDY_TITLE":"Trace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through ceramides (part III)","STUDY_SUMMARY":"Trace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through ceramides","INSTITUTE":"Mayo Clinic","LAST_NAME":"Lunt","FIRST_NAME":"Sophia","ADDRESS":"Michigan State University 410B Biochemistry Building 603 Wilson Road East Lansing, MI 48824","EMAIL":"sophia@msu.edu","PHONE":"517-432-4886"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"ms6138-1",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-2",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-3",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-4",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-5",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-6",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-7",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-8",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-9",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-10",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-11",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-12",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-13",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-14",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-15",
"Factors":{"experiment":"PSAT A13 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-16",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-17",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-18",
"Factors":{"experiment":"PSAT A13 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSAT"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-19",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-20",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-21",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-22",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-23",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-24",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-25",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-26",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-27",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-28",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-29",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-30",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-31",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-32",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-33",
"Factors":{"experiment":"PHGDH B34 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-34",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-35",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-36",
"Factors":{"experiment":"PHGDH B34 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PHGDH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-37",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-38",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-39",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-40",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-41",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-42",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"No label","time":"0 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-43",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-44",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-45",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-46",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-47",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-48",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glucose","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-49",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-50",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-51",
"Factors":{"experiment":"PSPH B8 + Dox (PKM1)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-52",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-53",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
},
{
"Subject ID":"-",
"Sample ID":"ms6138-54",
"Factors":{"experiment":"PSPH B8 - Dox (PKM2)","tracer":"13C-glutamine","time":"48 hr","group ID":"PSPH"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Pancreatic ductal adenocarcinoma cell line with PHGDH, PSAT, and PSPH KO. These cells have been incubated with no label, 13C-glucose or 13C-glutamine label for 48 hours."},

"TREATMENT":{"TREATMENT_SUMMARY":"Pancreatic cancer cells exhibit altered metabolism, which is mediated in part by expressing the proliferation-supportive M2 isoform of pyruvate kinase (PK). Unlike normal embryonic cells that stop proliferating when forced to express the proliferation-incompatible M1 isoform of PK, pancreatic cancer cells can proliferate just as rapidly with either the M1 or M2 isoform. During forced PKM1 expression, pancreatic cancer cells upregulate their serine biosynthesis pathway. The three enzymes in the serine biosynthesis pathway include phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSPH). Each of the genes encoding the three enzymes in the serine biosynthesis pathway have successfully been knocked out from pancreatic cancer cells using the CRISPR/Cas9 system in our laboratory, with multiple confirmed clones for each knockout ready for metabolic characterization."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"In this proposal, we will interrogate the metabolic pathways that support pancreatic cancer proliferation by performing isotope enrichment analysis on the CRISPR/Cas9 knockout pancreatic cancer cell lines. Each cell line expressing PKM1 or PKM2 will be seeded on 6-well plates so that they are ~70% confluent at the time of labeled media addition. Unlabeled media will be aspirated, and cell will be washed with PBS. Media labeled with 13C-glucose, 13C-glutamine, or 13C-serine labeled media will be added, and cells will be incubated for 1 hour or 24 hours. Polar metabolites and fatty acids will be extracted at the end of the incubation period using methanol, water, and chloroform. The methanol/water fraction containing polar metabolites will be separated from the chloroform fraction containing fatty acids, and each fraction will be dried down under nitrogen. Dried down samples will be sent to the Mayo Clinic Metabolomics Resource Core for analysis of TCA cycle intermediates, amino metabolites, free fatty acids, and sphingolipids. TCA cycle intermediates and amino metabolites (polar metabolites) should become labeled faster than fatty acids and sphingolipids; therefore, polar metabolites are proposed to be analyzed at 1 hour and 24 hours after 13C-label addition. Fatty acids are proposed to be analyzed for the 24 hr time point only, due to slow labeling. Statistical analysis will be performed by the Mayo Clinic Metabolomics Resource Core."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity BEH C8 (150 x 2mm, 1.7um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
"Units":"% enrichment: MPE",

"Data":[{"Metabolite":"Sphingosine 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}

}