Evers Maria, Song Jingwen, Shriwas Pratik, Greenbaum Harrison S, Chen Xiaozhuo
Honors Tutorial College, Ohio University, Athens, OH, United States.
The Ben May Department for Cancer Research, University of Chicago, Chicago, IL, United States.
Front Oncol. 2022 Jun 30;12:912065. doi: 10.3389/fonc.2022.912065. eCollection 2022.
We and others previously showed that extracellular ATP (eATP) is implicated in epithelial mesenchymal transition (EMT). However, the mechanisms by which eATP induces EMT and ATP's relationship to TGF-β, a well-known EMT inducer, are largely unclear. Also, eATP-induced EMT has never been studied at transcriptomic and metabolomics levels. Based on our previous studies, we hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels in cancer cells. RNAseq and metabolomics analyses were performed on human non-small cell lung cancer (NSCLC) A549 cells treated with either eATP or TGF-β. Bio-functional assays, such as invasion, intracellular ATP, cell proliferation, cytoskeleton remodeling, and others were conducted in NSCLC A549 and H1299 cells to validate changes observed from RNAseq and metabolomics studies. In the RNAseq study, eATP significantly enriched expressions of genes involved in EMT similarly to TGF-β after 2 and 6 hours of treatment. Samples treated with eATP for 2 hours share 131 upregulated EMT genes with those of TGF-β treated samples, and 42 genes at 6 hours treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. , one of the 11 genes, was selected for further study. eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar to those induced by TGF-β. Functional bioassays verified the findings from RNAseq and metabolomics that eATP EMT-like changes in A549 and H1299 cells similarly to TGF-β. was found to be implicated in EMT. In these studies, eATP-induced EMT, at all levels examined, is similar but non-identical to that induced by TGF-β, and functions in such a way that exogenous addition of TGF-β is unnecessary for the induction. The study of further verified its potential roles in EMT and the RNAseq analysis results. All these strongly indicate that eATP is a multi-functional and multi-locational inducer and regulator of EMT, changing our thinking on how EMT is induced and regulated and pointing to new directions for inhibiting EMT in cancer.
我们和其他研究人员之前表明，细胞外ATP（eATP）与上皮-间质转化（EMT）有关。然而，eATP诱导EMT的机制以及ATP与众所周知的EMT诱导剂转化生长因子-β（TGF-β）的关系在很大程度上尚不清楚。此外，从未在转录组学和代谢组学水平上研究过eATP诱导的EMT。基于我们之前的研究，我们假设eATP在癌细胞的各个水平上作为EMT的特异性诱导剂和调节剂发挥作用。对用eATP或TGF-β处理的人非小细胞肺癌（NSCLC）A549细胞进行了RNA测序和代谢组学分析。在NSCLC A549和H1299细胞中进行了生物功能测定，如侵袭、细胞内ATP、细胞增殖、细胞骨架重塑等，以验证从RNA测序和代谢组学研究中观察到的变化。在RNA测序研究中，处理2小时和6小时后，eATP与TGF-β类似，显著富集了参与EMT的基因表达。用eATP处理2小时的样本与用TGF-β处理的样本有131个上调的EMT基因相同，处理6小时时有42个基因相同。已经确定，在两个时间点，两种诱导剂均显著上调了11个在EMT中具有已知或未知功能的基因。其中一个基因被选作进一步研究。eATP在代谢途径中诱导了许多与EMT相关的变化，包括细胞骨架重排、糖酵解、谷氨酰胺分解、活性氧以及与TGF-β诱导的类似的个体代谢变化。功能生物测定验证了RNA测序和代谢组学的结果，即eATP在A549和H1299细胞中诱导了与TGF-β类似的EMT样变化。发现该基因与EMT有关。在这些研究中，在所有检测水平上，eATP诱导的EMT与TGF-β诱导的EMT相似但不完全相同，其作用方式是诱导过程中无需外源添加TGF-β。对该基因的研究进一步验证了其在EMT中的潜在作用以及RNA测序分析结果。所有这些都有力地表明，eATP是EMT的多功能、多位置诱导剂和调节剂，改变了我们对EMT如何被诱导和调节的认识，并为抑制癌症中的EMT指明了新方向。
