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.

中文翻译：

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蛋白质合成抑制剂通过驱动葡萄糖 6-磷酸的积累来刺激 MondoA 的转录活性

背景 蛋白质合成受氨基酸的可用性、生长因子信号通路的参与以及足以支持翻译的三磷酸腺苷 (ATP) 水平的调节。这些输入之间的串扰是广泛的，但其他调节机制仍有待表征。例如，翻译起始抑制剂 rocaglamide A (RocA) 诱导硫氧还蛋白相互作用蛋白 (TXNIP)。TXNIP 是葡萄糖摄取的负调节剂；因此，它由 RocA 诱导将翻译与葡萄糖的可用性联系起来。MondoA 是葡萄糖诱导转录的主要调节因子，其活性由糖酵解中间体葡萄糖 6-磷酸 (G6P) 触发。MondoA 响应由细胞质葡萄糖和线粒体 ATP (mtATP) 产生的 G6P，表明在细胞对这些能源的反应中起关键作用。TXNIP 表达完全依赖于 MondoA；因此，我们研究了蛋白质合成抑制剂如何影响其转录活性。方法 我们使用细胞系模型和功能丧失方法研究了翻译如何调节 MondoA 活性。我们分别使用 RNA 测序和代谢组学研究了蛋白质合成抑制剂如何影响基因表达和代谢。使用细胞系和患者来源的异种移植类器官 (PDxO) 模型评估了 RocA 的生物学影响。结果我们发现包括 RocA 在内的多种蛋白质合成抑制剂以依赖 MondoA、功能性电子传递链和 mtATP 合成的方式增加 TXNIP 表达。此外，RocA 和放线菌酮分别增加 mtATP 和 G6P 水平，TXNIP 的诱导取决于电压依赖性阴离子通道 (VDAC) 和生成 G6P 的己糖激酶 (HK) 之间的相互作用。RocA 处理影响约 1200 个基因的调节，其中约 250 个基因依赖于 MondoA。RocA 治疗对三阴性乳腺癌 (TNBC) 细胞系具有细胞毒性，并显示出对雌激素受体阴性 (ER-) PDxO 乳腺癌模型的优先细胞毒性。最后，RocA 驱动的细胞毒性部分依赖于 MondoA 或 TXNIP。结论我们的数据表明蛋白质合成抑制剂重新连接代谢，导致 mtATP 和 G6P 增加，后者驱动 MondoA 依赖性转录活性。此外，MondoA 是对 RocA 的细胞转录反应的关键组成部分。