Summary of Study ST000644
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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000644 |
| 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 |
| sophia@msu.edu | |
| Phone | 517-432-4886 |
| Submit Date | 2017-06-23 |
| Analysis Type Detail | LC-MS |
| Release Date | 2019-07-17 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU000667 |
| 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 | group ID |
|---|---|---|---|---|---|
| SA036385 | ms6138-26 | PHGDH B34 + Dox (PKM1) | 13C-glucose | 48 hr | PHGDH |
| SA036386 | ms6138-25 | PHGDH B34 + Dox (PKM1) | 13C-glucose | 48 hr | PHGDH |
| SA036387 | ms6138-27 | PHGDH B34 + Dox (PKM1) | 13C-glucose | 48 hr | PHGDH |
| SA036388 | ms6138-31 | PHGDH B34 + Dox (PKM1) | 13C-glutamine | 48 hr | PHGDH |
| SA036389 | ms6138-33 | PHGDH B34 + Dox (PKM1) | 13C-glutamine | 48 hr | PHGDH |
| SA036390 | ms6138-32 | PHGDH B34 + Dox (PKM1) | 13C-glutamine | 48 hr | PHGDH |
| SA036391 | ms6138-21 | PHGDH B34 + Dox (PKM1) | No label | 0 hr | PHGDH |
| SA036392 | ms6138-20 | PHGDH B34 + Dox (PKM1) | No label | 0 hr | PHGDH |
| SA036393 | ms6138-19 | PHGDH B34 + Dox (PKM1) | No label | 0 hr | PHGDH |
| SA036394 | ms6138-30 | PHGDH B34 - Dox (PKM2) | 13C-glucose | 48 hr | PHGDH |
| SA036395 | ms6138-29 | PHGDH B34 - Dox (PKM2) | 13C-glucose | 48 hr | PHGDH |
| SA036396 | ms6138-28 | PHGDH B34 - Dox (PKM2) | 13C-glucose | 48 hr | PHGDH |
| SA036397 | ms6138-36 | PHGDH B34 - Dox (PKM2) | 13C-glutamine | 48 hr | PHGDH |
| SA036398 | ms6138-35 | PHGDH B34 - Dox (PKM2) | 13C-glutamine | 48 hr | PHGDH |
| SA036399 | ms6138-34 | PHGDH B34 - Dox (PKM2) | 13C-glutamine | 48 hr | PHGDH |
| SA036400 | ms6138-24 | PHGDH B34 - Dox (PKM2) | No label | 0 hr | PHGDH |
| SA036401 | ms6138-22 | PHGDH B34 - Dox (PKM2) | No label | 0 hr | PHGDH |
| SA036402 | ms6138-23 | PHGDH B34 - Dox (PKM2) | No label | 0 hr | PHGDH |
| SA036403 | ms6138-9 | PSAT A13 + Dox (PKM1) | 13C-glucose | 48 hr | PSAT |
| SA036404 | ms6138-8 | PSAT A13 + Dox (PKM1) | 13C-glucose | 48 hr | PSAT |
| SA036405 | ms6138-7 | PSAT A13 + Dox (PKM1) | 13C-glucose | 48 hr | PSAT |
| SA036406 | ms6138-14 | PSAT A13 + Dox (PKM1) | 13C-glutamine | 48 hr | PSAT |
| SA036407 | ms6138-15 | PSAT A13 + Dox (PKM1) | 13C-glutamine | 48 hr | PSAT |
| SA036408 | ms6138-13 | PSAT A13 + Dox (PKM1) | 13C-glutamine | 48 hr | PSAT |
| SA036409 | ms6138-2 | PSAT A13 + Dox (PKM1) | No label | 0 hr | PSAT |
| SA036410 | ms6138-3 | PSAT A13 + Dox (PKM1) | No label | 0 hr | PSAT |
| SA036411 | ms6138-1 | PSAT A13 + Dox (PKM1) | No label | 0 hr | PSAT |
| SA036412 | ms6138-10 | PSAT A13 - Dox (PKM2) | 13C-glucose | 48 hr | PSAT |
| SA036413 | ms6138-12 | PSAT A13 - Dox (PKM2) | 13C-glucose | 48 hr | PSAT |
| SA036414 | ms6138-11 | PSAT A13 - Dox (PKM2) | 13C-glucose | 48 hr | PSAT |
| SA036415 | ms6138-16 | PSAT A13 - Dox (PKM2) | 13C-glutamine | 48 hr | PSAT |
| SA036416 | ms6138-17 | PSAT A13 - Dox (PKM2) | 13C-glutamine | 48 hr | PSAT |
| SA036417 | ms6138-18 | PSAT A13 - Dox (PKM2) | 13C-glutamine | 48 hr | PSAT |
| SA036418 | ms6138-5 | PSAT A13 - Dox (PKM2) | No label | 0 hr | PSAT |
| SA036419 | ms6138-6 | PSAT A13 - Dox (PKM2) | No label | 0 hr | PSAT |
| SA036420 | ms6138-4 | PSAT A13 - Dox (PKM2) | No label | 0 hr | PSAT |
| SA036421 | ms6138-44 | PSPH B8 + Dox (PKM1) | 13C-glucose | 48 hr | PSPH |
| SA036422 | ms6138-43 | PSPH B8 + Dox (PKM1) | 13C-glucose | 48 hr | PSPH |
| SA036423 | ms6138-45 | PSPH B8 + Dox (PKM1) | 13C-glucose | 48 hr | PSPH |
| SA036424 | ms6138-49 | PSPH B8 + Dox (PKM1) | 13C-glutamine | 48 hr | PSPH |
| SA036425 | ms6138-51 | PSPH B8 + Dox (PKM1) | 13C-glutamine | 48 hr | PSPH |
| SA036426 | ms6138-50 | PSPH B8 + Dox (PKM1) | 13C-glutamine | 48 hr | PSPH |
| SA036427 | ms6138-38 | PSPH B8 + Dox (PKM1) | No label | 0 hr | PSPH |
| SA036428 | ms6138-37 | PSPH B8 + Dox (PKM1) | No label | 0 hr | PSPH |
| SA036429 | ms6138-39 | PSPH B8 + Dox (PKM1) | No label | 0 hr | PSPH |
| SA036430 | ms6138-48 | PSPH B8 - Dox (PKM2) | 13C-glucose | 48 hr | PSPH |
| SA036431 | ms6138-46 | PSPH B8 - Dox (PKM2) | 13C-glucose | 48 hr | PSPH |
| SA036432 | ms6138-47 | PSPH B8 - Dox (PKM2) | 13C-glucose | 48 hr | PSPH |
| SA036433 | ms6138-52 | PSPH B8 - Dox (PKM2) | 13C-glutamine | 48 hr | PSPH |
| SA036434 | ms6138-53 | PSPH B8 - Dox (PKM2) | 13C-glutamine | 48 hr | PSPH |
| SA036435 | ms6138-54 | PSPH B8 - Dox (PKM2) | 13C-glutamine | 48 hr | PSPH |
| SA036436 | ms6138-40 | PSPH B8 - Dox (PKM2) | No label | 0 hr | PSPH |
| SA036437 | ms6138-41 | PSPH B8 - Dox (PKM2) | No label | 0 hr | PSPH |
| SA036438 | ms6138-42 | PSPH B8 - Dox (PKM2) | No label | 0 hr | PSPH |
| Showing results 1 to 54 of 54 |
Collection:
| Collection ID: | CO000661 |
| 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: | TR000681 |
| 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: | SP000674 |
| 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 | AN000976 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters Acquity BEH C8 (150 x 2mm,1.7um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6550 QTOF |
| Ion Mode | POSITIVE |
| Units | % enrichment: MPE |
Chromatography:
| Chromatography ID: | CH000701 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C8 (150 x 2mm,1.7um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000871 |
| Analysis ID: | AN000976 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
