Abstract

Mitochondrial glutamate-oxaloacetate (GOT2) is part of the malate-aspartate shuttle (MAS), a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the tumor microenvironment (TME).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting Source Data file; raw data are provided for metabolomics experiments, separated by tabs, in Supplemental Table 1.

Article and author information

Author details

  1. Samuel Kerk

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9786-2245
  2. Lin Lin

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  3. Amy L Myers

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  4. Damien J Sutton

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  5. Anthony Andren

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  6. Peter Sajjakulnukit

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  7. Li Zhang

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  8. Yaqing Zhang

    Department of Surgery, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  9. Jennifer A Jiménez

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  10. Barbara S Nelson

    Department of Surgery, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  11. Brandon Chen

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  12. Anthony Robinson

    Department of Cell and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  13. Galloway Thurston

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  14. Samantha B Kemp

    Molecular and Cellular Pathology Graduate Program, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  15. Nina G Steele

    Department of Cell and Developmental Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  16. Megan T Hoffman

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  17. Hui-Ju Wen

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  18. Daniel Long

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  19. Sarah E Ackenhusen

    Program in Chemical Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  20. Johanna Ramos

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  21. Xiaohua Gao

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  22. Zeribe C Nwosu

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  23. Stefanie Galban

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  24. Christopher J Halbrook

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
  25. David B Lombard

    Department of Pathology, University of Miami, Miami, United States
    Competing interests
    No competing interests declared.
  26. David R Piwnica-Worms

    Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, United States
    Competing interests
    No competing interests declared.
  27. Haoqiang Ying

    Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, United States
    Competing interests
    No competing interests declared.
  28. Marina Pasca di Magliano

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9632-9035
  29. Howard C Crawford

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    For correspondence
    hcrawfo1@hfhs.org
    Competing interests
    No competing interests declared.
  30. Yatrik M Shah

    Department of Molecular and Integrative Physiology, University of Michigan-Ann Arbor, Ann Arbor, United States
    For correspondence
    shahy@umich.edu
    Competing interests
    No competing interests declared.
  31. Costas A Lyssiotis

    Doctoral Program in Cancer Biology, University of Michigan-Ann Arbor, Ann Arbor, United States
    For correspondence
    clyssiot@umich.edu
    Competing interests
    Costas A Lyssiotis, has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics, and is an inventor on patents pertaining to Kras regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1-pathway as a therapeutic approach (US Patent No: 2015126580-A1, 05/07/2015; US Patent No: 20190136238, 05/09/2019; International Patent No: WO2013177426-A2, 04/23/2015)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9309-6141

Funding

National Institute of Allergy and Infectious Diseases (T32AI007413)

  • Samuel Kerk

National Cancer Institute (R37CA237421)

  • Costas A Lyssiotis

National Cancer Institute (R01CA248160)

  • Costas A Lyssiotis

National Cancer Institute (R01CA244931)

  • Costas A Lyssiotis

National Cancer Institute (F31CA254079)

  • Jennifer A Jiménez

National Institute of Diabetes and Digestive and Kidney Diseases (T32-DK094775)

  • Barbara S Nelson

National Cancer Institute (T32-CA009676)

  • Barbara S Nelson

National Cancer Institute (R50 CA232985)

  • Yaqing Zhang

National Institute of General Medical Sciences (T32-GM11390)

  • Samantha B Kemp

National Cancer Institute (F31-CA247076)

  • Samantha B Kemp

National Cancer Institute (T32-CA009676)

  • Nina G Steele

National Cancer Institute (F31CA24745701)

  • Samuel Kerk

American Cancer Society (PF-19-096-01)

  • Nina G Steele

National Cancer Institute (K99CA241357)

  • Christopher J Halbrook

National Institute of Diabetes and Digestive and Kidney Diseases (P30DK034933)

  • Christopher J Halbrook

National Institute of General Medical Sciences (R01GM101171)

  • David B Lombard

National Cancer Institute (CA253986)

  • David B Lombard

National Cancer Center (P30 CA046592)

  • Costas A Lyssiotis

National Cancer Institute (1F99CA264414-01)

  • Samuel Kerk

National Cancer Institute (CA148828)

  • Yatrik M Shah

National Cancer Institute (CA245546)

  • Yatrik M Shah

Pancreatic Cancer Action Network (13-70-25-LYSS)

  • Costas A Lyssiotis

V Foundation for Cancer Research (V2016-009)

  • Costas A Lyssiotis

Sidney Kimmel Foundation (SKF-16-005)

  • Costas A Lyssiotis

American Association for Cancer Research (17-20-01-LYSS)

  • Costas A Lyssiotis

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Lydia W S Finley, Memorial Sloan Kettering Cancer Center, United States

Ethics

Animal experimentation: Animal experiments were conducted in accordance with the Office of Laboratory Animal Welfare and approved by the Institutional Animal Care and Use Committees of the University of Michigan. ULAM: PRO00008877

Version history

  1. Preprint posted: August 7, 2020 (view preprint)
  2. Received: August 21, 2021
  3. Accepted: July 9, 2022
  4. Accepted Manuscript published: July 11, 2022 (version 1)
  5. Version of Record published: July 27, 2022 (version 2)

Copyright

© 2022, Kerk et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Samuel Kerk
  2. Lin Lin
  3. Amy L Myers
  4. Damien J Sutton
  5. Anthony Andren
  6. Peter Sajjakulnukit
  7. Li Zhang
  8. Yaqing Zhang
  9. Jennifer A Jiménez
  10. Barbara S Nelson
  11. Brandon Chen
  12. Anthony Robinson
  13. Galloway Thurston
  14. Samantha B Kemp
  15. Nina G Steele
  16. Megan T Hoffman
  17. Hui-Ju Wen
  18. Daniel Long
  19. Sarah E Ackenhusen
  20. Johanna Ramos
  21. Xiaohua Gao
  22. Zeribe C Nwosu
  23. Stefanie Galban
  24. Christopher J Halbrook
  25. David B Lombard
  26. David R Piwnica-Worms
  27. Haoqiang Ying
  28. Marina Pasca di Magliano
  29. Howard C Crawford
  30. Yatrik M Shah
  31. Costas A Lyssiotis
(2022)
Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context
eLife 11:e73245.
https://doi.org/10.7554/eLife.73245

Share this article

https://doi.org/10.7554/eLife.73245

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