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Updated: Nov 26, 2025
Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Published on: October 23, 2018
Protein synthesis inhibitors stimulate MondoA transcriptional activity by driving an accumulation of glucose
Blake R Wilde1,2, Mohan R Kaadige1,3, Katrin P Guillen4
1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112, USA.
Background:
Protein synthesis is regulated by the availability of amino acids, the engagement of growth factor signaling pathways, and adenosine triphosphate (ATP) levels sufficient to support translation. Crosstalk between these inputs is extensive, yet other regulatory mechanisms remain to be characterized. For example, the translation initiation inhibitor rocaglamide A (RocA) induces thioredoxin-interacting protein (TXNIP). TXNIP is a negative regulator of glucose uptake; thus, its induction by RocA links translation to the availability of glucose. MondoA is the principal regulator of glucose-induced transcription, and its activity is triggered by the glycolytic intermediate, glucose 6-phosphate (G6P). MondoA responds to G6P generated by cytoplasmic glucose and mitochondrial ATP (mtATP), suggesting a critical role in the cellular response to these energy sources. TXNIP expression is entirely dependent on MondoA; therefore, we investigated how protein synthesis inhibitors impact its transcriptional activity.
Methods:
We investigated how translation regulates MondoA activity using cell line models and loss-of-function approaches. We examined how protein synthesis inhibitors effect gene expression and metabolism using RNA-sequencing and metabolomics, respectively. The biological impact of RocA was evaluated using cell lines and patient-derived xenograft organoid (PDxO) models.
Results:
We discovered that multiple protein synthesis inhibitors, including RocA, increase TXNIP expression in a manner that depends on MondoA, a functional electron transport chain and mtATP synthesis. Furthermore, RocA and cycloheximide increase mtATP and G6P levels, respectively, and TXNIP induction depends on interactions between the voltage-dependent anion channel (VDAC) and hexokinase (HK), which generates G6P. RocA treatment impacts the regulation of ~ 1200 genes, and ~ 250 of those genes are MondoA-dependent. RocA treatment is cytotoxic to triple negative breast cancer (TNBC) cell lines and shows preferential cytotoxicity against estrogen receptor negative (ER-) PDxO breast cancer models. Finally, RocA-driven cytotoxicity is partially dependent on MondoA or TXNIP.
Conclusions:
Our data suggest that protein synthesis inhibitors rewire metabolism, resulting in an increase in mtATP and G6P, the latter driving MondoA-dependent transcriptional activity. Further, MondoA is a critical component of the cellular transcriptional response to RocA. Our functional assays suggest that RocA or similar translation inhibitors may show efficacy against ER- breast tumors and that the levels of MondoA and TXNIP should be considered when exploring these potential treatment options.
Insights
Protein synthesis inhibitors like rocaglamide A (RocA) increase mitochondrial ATP and glucose 6-phosphate, activating MondoA-dependent transcription. This rewiring of metabolism shows potential efficacy against estrogen receptor-negative breast tumors.
Area of Science:
- Molecular Biology
- Cellular Metabolism
- Cancer Therapeutics
Background:
- Protein synthesis is regulated by amino acids, growth factors, and ATP, with extensive crosstalk between inputs.
- Rocaglamide A (RocA), a translation inhibitor, induces thioredoxin-interacting protein (TXNIP), linking translation to glucose availability.
- MondoA, a key regulator of glucose transcription, responds to glucose 6-phosphate (G6P) and mitochondrial ATP (mtATP).
Purpose of the Study:
- To investigate how protein synthesis inhibitors impact MondoA's transcriptional activity and its link to cellular metabolism.
- To elucidate the role of MondoA and TXNIP in the cellular response to RocA.
- To evaluate the therapeutic potential of RocA against specific breast cancer subtypes.
Main Methods:
- Utilized cell line models and loss-of-function approaches to study translation's regulation of MondoA activity.
- Employed RNA-sequencing and metabolomics to analyze gene expression and metabolic changes induced by protein synthesis inhibitors.
- Assessed the biological impact and cytotoxicity of RocA using cell lines and patient-derived xenograft organoid (PDxO) models.
Main Results:
- Protein synthesis inhibitors, including RocA, elevate TXNIP expression dependent on MondoA, electron transport chain, and mtATP synthesis.
- RocA and cycloheximide increase mtATP and G6P, with TXNIP induction requiring interactions between VDAC and hexokinase (HK).
- RocA treatment exhibits cytotoxicity against triple-negative breast cancer (TNBC) and estrogen receptor-negative (ER-) PDxO models, partially dependent on MondoA or TXNIP.
Conclusions:
- Protein synthesis inhibitors rewire cellular metabolism, increasing mtATP and G6P, which drives MondoA-dependent transcription.
- MondoA is crucial for the transcriptional response to RocA, and its interplay with TXNIP is vital.
- RocA and similar inhibitors may be effective against ER- breast tumors, with MondoA and TXNIP levels being important considerations for treatment.
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