Summary of Study ST000642

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 PR000459. The data can be accessed directly via it's Project DOI: 10.21228/M8QP56 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000642
Study TitleTrace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through free fatty acids (part I)
Study SummaryTrace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through free fatty acids
Institute
Mayo Clinic
Last NameLunt
First NameSophia
AddressMichigan State University 410B Biochemistry Building 603 Wilson Road East Lansing, MI 48824
Emailsophia@msu.edu
Phone517-432-4886
Submit Date2017-06-23
Analysis Type DetailLC-MS
Release Date2019-07-17
Release Version1
Sophia Lunt Sophia Lunt
https://dx.doi.org/10.21228/M8QP56
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000459
Project DOI:doi: 10.21228/M8QP56
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

Subject:

Subject ID:SU000665
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

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

mb_sample_id local_sample_id experiment tracer time
SA036313ms5770-120_PSPH B7 + Dox (PKM1)_0 hr_No label No Label -
SA036314ms5770-221_PSPH B7 + Dox (PKM1)_0 hr_No label No Label -
SA036315ms5770-322_PSPH B7 + Dox (PKM1)_0 hr_No label No Label -
SA036316ms5770-423_PSPH B7 - Dox (PKM2)_0 hr_No label No Label -
SA036317ms5770-524_PSPH B7 - Dox (PKM2)_0 hr_No label No Label -
SA036318ms5770-625_PSPH B7 - Dox (PKM2)_0 hr_No label No Label -
SA036319ms5770-726_PSPH B7 + Dox (PKM1)_24 hr_13C-glucose 13C-glucose 24
SA036320ms5770-827_PSPH B7 + Dox (PKM1)_24 hr_13C-glucose 13C-glucose 24
SA036321ms5770-928_PSPH B7 + Dox (PKM1)_24 hr_13C-glucose 13C-glucose 24
SA036322ms5770-1029_PSPH B7 - Dox (PKM2)_24 hr_13C-glucose 13C-glucose 24
SA036323ms5770-1130_PSPH B7 - Dox (PKM2)_24 hr_13C-glucose 13C-glucose 24
SA036324ms5770-1231_PSPH B7 - Dox (PKM2)_24 hr_13C-glucose 13C-glucose 24
SA036325ms5770-1332_PSPH B7 + Dox (PKM1)_48 hr_13C-glucose 13C-glucose 48
SA036326ms5770-1433_PSPH B7 + Dox (PKM1)_48 hr_13C-glucose 13C-glucose 48
SA036327ms5770-1534_PSPH B7 + Dox (PKM1)_48 hr_13C-glucose 13C-glucose 48
SA036328ms5770-1635_PSPH B7 - Dox (PKM2)_48 hr_13C-glucose 13C-glucose 48
SA036329ms5770-1736_PSPH B7 - Dox (PKM2)_48 hr_13C-glucose 13C-glucose 48
SA036330ms5770-1837_PSPH B7 - Dox (PKM2)_48 hr_13C-glucose 13C-glucose 48
Showing results 1 to 18 of 18

Collection:

Collection ID:CO000659
Collection Summary:Pancreatic ductal adenocarcinoma cell line with PSPH KO. These cells have been incubated with no label, or 13C-glucose label for 24hours or 48 hours.
Sample Type:Pancreas

Treatment:

Treatment ID:TR000679
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.

Sample Preparation:

Sampleprep ID:SP000672
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.

Combined analysis:

Analysis ID AN000974
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Thermo Quantum Ultra
Ion Mode POSITIVE
Units % enrichment: MPE

Chromatography:

Chromatography ID:CH000699
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS000869
Analysis ID:AN000974
Instrument Name:Thermo Quantum Ultra
Instrument Type:Triple quadrupole
MS Type:ESI
Ion Mode:POSITIVE
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