The Pancreatic Tumor Microenvironment Compensates for Loss of GOT2

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The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and, consequently, access to blood-derived nutrients and oxygen, which impacts tumor growth. Intracellular redox imbalance is another restraint on cellular proliferation, yet it is unknown if the TME contributes to the maintenance of redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle, disturbs redox homeostasis and halts proliferation of PDA cells in vitro. In contrast, GOT2 inhibition has no effect on in vivo tumor growth or tumorigenesis in an autochthonous model. We propose that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts. More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Genetic or pharmacologic inhibition of pyruvate uptake or metabolism abrogated pyruvate-mediated alleviation of reductive stress from NADH buildup. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.

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  1. Evaluation Summary:

    This paper investigating specific metabolic dependencies of pancreatic cancer cells growing in vitro and in vivo will be of interest to scientists in the field of cancer metabolism. The data reveal that cancer-associated stromal cells can play an important role supporting the redox state of cancer cells cultured in vitro, but at present the data do not support the conclusion that this mechanism controls the metabolic resilience of cancer cells growing in vivo and alternate hypotheses remain to be addressed.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    In this manuscript, the authors investigate the role of glutamate-oxaloacetate transaminase 2 (GOT2) in the growth of pancreatic ductal adenocarcinoma (PDAC). Building on previous work demonstrating an important role of cytosolic malic enzyme in PDAC progression, the authors tested the importance of GOT2, an enzyme in the malate-aspartate shuttle, as a therapeutic target. The authors find that GOT2 is required for colony formation of PDAC cells in vitro but is entirely dispensable for growth of both xenograft and autochthonous models of PDAC in mice. A major strength of the manuscript is the multitude of cell lines used, demonstrating the generalizability of these findings in vitro, as well as the generation of novel mouse models for GOT2 perturbation.

    In vitro, GOT2 deficiency can be rescued by conditioned medium from cancer associated fibroblasts (CAF) or exogenous pyruvate. Mechanistically, pyruvate serves to alter redox balance that rescues GOT2 loss. In vitro, perturbing pyruvate import or metabolism does not affect tumor growth. The finding that GOT2 deficiency can be rescued by restoring cytosolic redox adds a new layer of understanding to the role of GOT2 in supporting cancer cell growth. The discovery that CAFs contribute to redox balance in cancer cells represents an important conceptual advance. However, whether maintenance of redox balance underlies the lack of phenotype of GOT2-deficient tumors in vitro remains unclear, as interventions to block pyruvate uptake or conversion to lactate have no impact on tumor growth. While technical issues may underlie these negative results, at present there is a lack of evidence supporting the conclusion that CAFs promote redox balance in vivo. Alternative explanations for the ability of GOT2-deficient tumors to sustain growth are not explored. Therefore, the manuscript at present does not resolve whether CAFs support cancer cell redox balance in vivo or, more broadly, whether redox balance is a constraint on cancer cell growth in vivo, is not addressed.

  3. Reviewer #2 (Public Review):

    Cancer cells frequently display changes to cell metabolism, suggesting that impairments in cancer specific metabolic dependencies may represent a viable path to improve cancer therapy. Work from these authors and others has determined that Kras mutant pancreatic cancer cells may alter the metabolism of TCA cycle associated metabolites and cause a particular dependence on malic enzyme 1 (ME1). The substrate of ME1, malate, is in relation to metabolites and metabolic enzymes in the malate-aspartate shuttle, including through the activity of mitochondrial transaminase enzyme GOT2. In this manuscript Kerk et al. investigate the role for GOT2 in support of pancreatic cancer cell proliferation in culture and in murine tumor models. The authors find that, despite GOT2 loss causing robust proliferation impairments in culture, the same alteration has no significant effect on tumor growth in vivo. Mechanistically, the authors determine that fibroblast conditioned media can cause resistance to GOT2 dependent proliferation defects in PDAC cells in culture, likely because of pyruvate released by fibroblasts that functions as an electron acceptor to promote cell proliferation. Regardless, no significant effect on tumor burden is observed in autochthonous mouse models of GOT2 deficient PDAC, nor does impairing pyruvate uptake synergize with GOT2 loss to prevent growth of PDAC xenografts.

    Overall, the manuscript is well done, with rigorous use of pancreatic cancer cell lines and mouse models, comprehensive metabolic profiling, and strong mechanistic investigations of the role of pyruvate supplementation in GOT2 knockdown cells, however, the conclusions are somewhat disappointing since no clear answer is reached as to how these cells proliferate without GOT2 in tumors. From a writing perspective, I commend the authors for being honest about these limitations and describing possible explanations in earnest. The conclusions reached by the authors are therefore justified by the data. That said, the manuscript ultimately serves as a cautionary tale that the metabolic dependencies identified in culture may not translate to the complex microenvironment of tumors. Nonetheless, the hypothesis was well founded, and the results are highly interpretable, making this manuscript of interest for the scientific community.

  4. Reviewer #3 (Public Review):

    This is a nicely done study and well written article by Kerk and colleagues focusing on the role of GOT2 in pancreatic tumor growth that highlights critical differences between in vitro and in vivo dependencies. Such differences are crucial to understand for effective targeting of metabolic pathways in PDA. The authors show that GOT2 silencing drastically impairs PDA cell colony formation in vitro, but that it is not required for PDA tumor growth in vivo. This led authors to hypothesize that tumor microenvironmental factors may facilitate growth in the absence of GOT2 in vivo. Authors go on to find that conditioned medium from fibroblasts is sufficient to rescue colony formation in GOT2 KD cells, that pyruvate accumulates in CM, and that pyruvate is also sufficient to rescue colony formation. It is shown that PDA cells convert pyruvate into lactate and that mitochondrial import of pyruvate is not required for the rescue. These data lead authors to test whether GOT2 loss disrupts redox homeostasis, finding an increase in the NADH/NAD+ ratio. Other manipulations that boost NAD+ production such as LbNox expression or aKB supplementation partially rescue colony formation. MCT1 KO or inhibition also impairs pyruvate or CM rescue in vitro, though does not impact tumor growth in vivo. Finally, authors generate KC-Got2 mice and show that they have no differences in tumor formation. Overall, the study is well done and conclusions appropriate, although additional controls are warranted. While disappointing that Got2 KO and MCT inhibition together had no effect in vivo, the findings highlight the challenges of targeting tumor metabolism and authors do a good job of discussing the relevant factors that may explain this result. I commend them for transparently reporting the negative in vivo data.