Summary of Study ST000643

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 IDST000643
Study TitleTrace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through non-esterified fatty acids (part II)
Study SummaryTrace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through non-esterified 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:SU000666
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 groupID
SA036331ms6129-26PHGDH B34 + Dox (PKM1) 13C-glucose 48 PHGDH
SA036332ms6129-25PHGDH B34 + Dox (PKM1) 13C-glucose 48 PHGDH
SA036333ms6129-27PHGDH B34 + Dox (PKM1) 13C-glucose 48 PHGDH
SA036334ms6129-31PHGDH B34 + Dox (PKM1) 13C-glutamine 48 PHGDH
SA036335ms6129-33PHGDH B34 + Dox (PKM1) 13C-glutamine 48 PHGDH
SA036336ms6129-32PHGDH B34 + Dox (PKM1) 13C-glutamine 48 PHGDH
SA036337ms6129-21PHGDH B34 + Dox (PKM1) No label - PHGDH
SA036338ms6129-20PHGDH B34 + Dox (PKM1) No label - PHGDH
SA036339ms6129-19PHGDH B34 + Dox (PKM1) No label - PHGDH
SA036340ms6129-30PHGDH B34 - Dox (PKM2) 13C-glucose 48 PHGDH
SA036341ms6129-29PHGDH B34 - Dox (PKM2) 13C-glucose 48 PHGDH
SA036342ms6129-28PHGDH B34 - Dox (PKM2) 13C-glucose 48 PHGDH
SA036343ms6129-36PHGDH B34 - Dox (PKM2) 13C-glutamine 48 PHGDH
SA036344ms6129-35PHGDH B34 - Dox (PKM2) 13C-glutamine 48 PHGDH
SA036345ms6129-34PHGDH B34 - Dox (PKM2) 13C-glutamine 48 PHGDH
SA036346ms6129-24PHGDH B34 - Dox (PKM2) No label - PHGDH
SA036347ms6129-22PHGDH B34 - Dox (PKM2) No label - PHGDH
SA036348ms6129-23PHGDH B34 - Dox (PKM2) No label - PHGDH
SA036349ms6129-9PSAT A13 + Dox (PKM1) 13C-glucose 48 PSAT
SA036350ms6129-8PSAT A13 + Dox (PKM1) 13C-glucose 48 PSAT
SA036351ms6129-7PSAT A13 + Dox (PKM1) 13C-glucose 48 PSAT
SA036352ms6129-14PSAT A13 + Dox (PKM1) 13C-glutamine 48 PSAT
SA036353ms6129-15PSAT A13 + Dox (PKM1) 13C-glutamine 48 PSAT
SA036354ms6129-13PSAT A13 + Dox (PKM1) 13C-glutamine 48 PSAT
SA036355ms6129-2PSAT A13 + Dox (PKM1) No label - PSAT
SA036356ms6129-3PSAT A13 + Dox (PKM1) No label - PSAT
SA036357ms6129-1PSAT A13 + Dox (PKM1) No label - PSAT
SA036358ms6129-10PSAT A13 - Dox (PKM2) 13C-glucose 48 PSAT
SA036359ms6129-12PSAT A13 - Dox (PKM2) 13C-glucose 48 PSAT
SA036360ms6129-11PSAT A13 - Dox (PKM2) 13C-glucose 48 PSAT
SA036361ms6129-16PSAT A13 - Dox (PKM2) 13C-glutamine 48 PSAT
SA036362ms6129-17PSAT A13 - Dox (PKM2) 13C-glutamine 48 PSAT
SA036363ms6129-18PSAT A13 - Dox (PKM2) 13C-glutamine 48 PSAT
SA036364ms6129-5PSAT A13 - Dox (PKM2) No label - PSAT
SA036365ms6129-6PSAT A13 - Dox (PKM2) No label - PSAT
SA036366ms6129-4PSAT A13 - Dox (PKM2) No label - PSAT
SA036367ms6129-44PSPH B8 + Dox (PKM1) 13C-glucose 48 PSPH
SA036368ms6129-43PSPH B8 + Dox (PKM1) 13C-glucose 48 PSPH
SA036369ms6129-45PSPH B8 + Dox (PKM1) 13C-glucose 48 PSPH
SA036370ms6129-49PSPH B8 + Dox (PKM1) 13C-glutamine 48 PSPH
SA036371ms6129-51PSPH B8 + Dox (PKM1) 13C-glutamine 48 PSPH
SA036372ms6129-50PSPH B8 + Dox (PKM1) 13C-glutamine 48 PSPH
SA036373ms6129-38PSPH B8 + Dox (PKM1) No label - PSPH
SA036374ms6129-37PSPH B8 + Dox (PKM1) No label - PSPH
SA036375ms6129-39PSPH B8 + Dox (PKM1) No label - PSPH
SA036376ms6129-48PSPH B8 - Dox (PKM2) 13C-glucose 48 PSPH
SA036377ms6129-46PSPH B8 - Dox (PKM2) 13C-glucose 48 PSPH
SA036378ms6129-47PSPH B8 - Dox (PKM2) 13C-glucose 48 PSPH
SA036379ms6129-52PSPH B8 - Dox (PKM2) 13C-glutamine 48 PSPH
SA036380ms6129-53PSPH B8 - Dox (PKM2) 13C-glutamine 48 PSPH
SA036381ms6129-54PSPH B8 - Dox (PKM2) 13C-glutamine 48 PSPH
SA036382ms6129-40PSPH B8 - Dox (PKM2) No label - PSPH
SA036383ms6129-41PSPH B8 - Dox (PKM2) No label - PSPH
SA036384ms6129-42PSPH B8 - Dox (PKM2) No label - PSPH
Showing results 1 to 54 of 54

Collection:

Collection ID:CO000660
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.
Sample Type:Pancreas

Treatment:

Treatment ID:TR000680
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:SP000673
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 AN000975
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 QTOF
MS instrument name Agilent 6550 QTOF
Ion Mode NEGATIVE
Units % enrichment: MPE

Chromatography:

Chromatography ID:CH000700
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (150 x 2.1mm, 1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS000870
Analysis ID:AN000975
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:NEGATIVE
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