Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context
- University of Michigan-Ann Arbor, United States
- University of Miami, United States
- The University of Texas MD Anderson Cancer Center, United States
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
Marina Pasca di Magliano
Costas A Lyssiotis
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.
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
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.
Metrics
-
- 4,080
- views
-
- 818
- downloads
-
- 43
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context
eLife 11:e73245.
https://doi.org/10.7554/eLife.73245
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
