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. 2020 Dec 4;8(1):27.
doi: 10.1186/s40170-020-00233-6.

Protein synthesis inhibitors stimulate MondoA transcriptional activity by driving an accumulation of glucose 6-phosphate

Blake R Wilde  1   2 , Mohan R Kaadige  1   3 , Katrin P Guillen  4 , Andrew Butterfield  4 , Bryan E Welm  4 , Donald E Ayer  5
Affiliations

Protein synthesis inhibitors stimulate MondoA transcriptional activity by driving an accumulation of glucose 6-phosphate

Blake R Wilde et al. Cancer Metab. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
TXNIP expression is suppressed by translation. a Heatmaps showing TXNIP mRNA relative to ribosomal protein L24 in the genotype-tissue expression project (GTEx) database. Spearman correlation statistics are reported. b, c TXNIP mRNA levels in HeLa cells following 16-h treatments with the translation elongation inhibitors CHX (50 μg/mL), emetine (50 μg/mL), and puromycin (100 μg/mL) or the translation initiation inhibitor Rocaglamide A (RocA, 100 nM). d Relative rate of 3H-2-deoxyglucose uptake in HeLa cells following 16-h treatments with RocA or vehicle. e, f TXNIP mRNA and protein levels of the indicated proteins in HeLa cells transfected with a pool of four siRNAs against EIF4E or a pool of four scrambled siRNA controls. g TXNIP mRNA levels in HeLa cells following 16-h treatments with CHX or Torin (250 nM). TXNIP mRNA levels following 16-h CHX treatments of h C2C12 mouse myoblasts, i L6 rat myoblasts, and j 293T embryonic kidney cells. TXNIP mRNA levels were determined using RT-qPCR
Fig. 2
Fig. 2
Protein synthesis inhibition drives MondoA transcriptional activity. TXNIP mRNA levels following 16-h CHX treatments of a MondoA+/+ and MondoA−/− MEFs, and b MondoA−/− MEFs expressing empty vector or MondoA. c Immunofluorescence was used to assess the subcellular localization of MondoA in HeLa cells treated with CHX for 16 h. Cells were scored for localization of MondoA (cytoplasmic > nuclear or cytoplasmic ≤ nuclear). d Chromatin immunoprecipitation was used to determine the enrichment of MondoA on the TXNIP promoter in HeLa cells treated with CHX for 16 h. e HeLa cells transfected with the indicated reporter luciferase constructs were treated with CHX for 16 h. The ChoREmut TXNIP promoter lacks the double CACGAG carbohydrate responsive element located directly upstream of luciferase. The media was replaced with regular medium for 1 h to wash out CHX, allowing translation of accumulated luciferase mRNA. f To ensure that TXNIP levels were at a minimum, HeLa cells were starved of glucose for 6 h prior to treatment with CHX. We then measured TXNIP mRNA levels in cells growing in DMEM +10% FBS or glucose-free DMEM +10% FBS following 16-h CHX treatments. g TXNIP mRNA levels in HeLa cells growing in glucose-free DMEM +10% FBS or in glucose-free DMEM +10% dialyzed FBS following 16-h treatments. h TXNIP mRNA levels in HeLa cells growing in glucose-free DMEM +10% dialyzed FBS with the indicated amount of glucose following 16-h treatments with CHX. TXNIP mRNA levels were determined using RT-qPCR
Fig. 3
Fig. 3
Protein synthesis inhibition drives G6P synthesis. a TXNIP mRNA levels in HeLa cells treated with CHX and the electron transport chain inhibitor metformin (Met, 5 mM) or oligomycin (Olig, 1 μM) for 16 h. b TXNIP mRNA levels following a 16-h RocA treatment of HeLa cells transfected with pool of siRNA specific for ATP5I (siATP5I) or a pool of scrambled control siRNAs (siSCRM). c A mitochondrial-targeted ATP FRET biosensor (mitATEAM) was used to determine relative mtATP levels in HeLa cells treated with the protein synthesis inhibitor RocA for up to 6 h. Relative mtATP was determined as the ratio of FRET to CFP intensities. d TXNIP mRNA levels following a 16-h RocA treatment (100 nM) of HeLa cells expressing wildtype mouse VDAC1 (mVDAC1) or VDAC1(E72Q), which cannot bind Hexokinase II. e TXNIP mRNA levels in HeLa cells treated for 16 h with CHX or methyl-jasmonate (3 mM). f Heatmap showing relative metabolite levels from HeLa cells treated with CHX for 16 h. Metabolite levels were assessed through GC-MS. f Log2(fold-change) of glycolytic and TCA cycle intermediates from HeLa cells treated with CHX for 16 h, relative to control DMSO treatment. TXNIP mRNA levels were determined using RT-qPCR
Fig. 4
Fig. 4
Protein synthesis inhibition drives TXNIP expression independent of oncogenic burden. TXNIP mRNA levels following a 16-h CHX treatment of a wildtype or HRAS(G12V)-expressing murine embryonic fibroblasts (MEFs), b TSC2−/− MEFs expressing empty vector or human-TSC2, and c MDA-MB-231 expressing tet-inducible MYC(T58A) with or without doxycyline. Immunoblots showing TXNIP, MondoA, and tubulin protein levels following 16-h RocA treatment of d HeLa cells, e MDA-MB-157 cells, and f MDA-MB-231 cells. TXNIP mRNA levels were determined using RT-qPCR
Fig. 5
Fig. 5
Cytotoxicity elicited by protein synthesis inhibitors requires TXNIP. a Relative cell viability over the indicated time course of MDA-MB-157 and MDA-MB-231 cells in the presence of RocA (100 nM) was assessed by crystal violet staining. b Viability of patient-derived xenograft organoids (PDxOs) following treatment with RocA at various concentrations. PDxOs are separated into ER+ and ER− groups. c TXNIP+/+ or TXNIP−/− MEFS were treated with RocA for 2 days; then, cell viability was analyzed using crystal violet staining. d MDA-MB-157 cells expressing scrambled shRNA (shScrm) or shTXNIP were treated with 100 nM RocA for 2 days; then, cell viability was analyzed using crystal violet staining. e We previously characterized HeLa cells in which MondoA was knocked out by CRISPR/Cas9 [13]. Cells were treated with RocA for 2 days, and then, cell viability was analyzed using crystal violet staining
Fig. 6
Fig. 6
The MondoA-dependent transcriptional response to translation inhibition. mRNA sequencing was used to determine gene expression changes in HeLa and HeLa:MondoA-KO cells following 4-h treatments with 100 nM RocA. a Heatmap depicting the top 500 genes regulated by RocA treatment. Regression analysis using DESeq2 was performed to generate a genotype:treatment interaction scores. b A volcano plot showing log2(fold-change) of HeLa cells treated with RocA compared to HeLa:MondoA-KO cells treated with RocA. Genes with an adjusted p value ≤ 1E−10 are indicated in blue (downregulated) and red (upregulated). c Overrepresentation analysis was used to determine pathways that are dysregulated in HeLa cells treated with RocA compared to HeLa:MondoA-KO cells treated with RocA. d Gene set enrichment analysis and leading-edge analysis was performed using all gene sets in the Molecular Signature Database (Broad Institute). HeLa cells treated with RocA were compared to HeLa:MondoA-KO cells treated with RocA. Nodes that contain at least 4 gene sets are shown
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