During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already “Warburg-like” context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.

中文翻译：

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在乳腺癌细胞的上皮间质转化过程中微调代谢重连和翻译机制的适应


在乳腺癌进展过程中，上皮细胞向间质细胞的转变与转移和内分泌治疗耐药有关。然而，根本机制仍然难以捉摸。为了深入了解这一过程，我们研究了 MCF7 衍生细胞所经历的转变，该转变是由缺乏肌动蛋白结合域 (MKL1 ΔN200) 的 MKL1 变体的组成型核表达驱动的。我们表征了 MKL1 诱导的细胞模型期间发生的适应性变化，并重点关注翻译机制和代谢适应的调节。我们使用核糖体分析与 RNA-Seq 相结合，在转录和翻译水平上进行了全基因组分析，并分析了翻译机制组件和参与能量代谢的酶的表达。 NGS 数据与从多核糖体和蛋白质印迹中提取的特定 mRNA 的代谢组学测量和定量相关。我们的结果揭示了根据上皮到间质转变的管腔到基底样状态的表达谱。在转变过程中，核糖体蛋白和许多翻译因子的合成上调。翻译机制的过度表达似乎在翻译水平上受到调节。我们的结果表明核糖体生物发生和翻译活性增加。我们检测到在已经“Warburg 样”的环境中发生了广泛的代谢重连，其中酶异构体转换和代谢分流表明 HIF-1α 以及其他主要调节因子的关键作用。此外，我们检测到参与戊糖磷酸途径的核糖核苷酸合成的酶的表达减少。 在此转变期间，细胞体积增大，下调与增殖相关的基因，并强烈上调细胞骨架和细胞外基质基因的表达。我们的研究揭示了上皮间充质样转变过程中与代谢和翻译机制适应相关的多种调控事件。在这一主要的细胞转变过程中，细胞达到新的稳态，确保其生存。这项工作表明，核糖体分析与 RNA-Seq 相结合是一种强大的方法，可以揭示深入的全局适应性细胞反应和调节回路之间的互连，这将有助于识别新的治疗靶点。