From transcriptomics to metabolomics: Extracellular ATP induces TGF-β-like and TGF-β-independent epithelial mesenchymal transition in lung cancer cells

Abstract Background: Epithelial mesenchymal transition (EMT) is an early process in metastasis. Extracellular ATP (eATP) was shown to play important roles in EMT. However, the mechanisms by which eATP induces EMT and ATP’s relationship to TGF-b, a well-known EMT inducer, are unclear. Key questions include: if and how much EMT-specific gene expression eATP induces and how similar is ATP-induced EMT to TGF-b-induced EMT? We hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels alternative to TGF-b in cancer cells. Methods: As EMT involves changes from gene expression to metabolites, RNAseq and metabolomics analyses were performed on human NSCLC A549 cells treated with either eATP or TGF-b to determine how they induce EMT at transcription and metabolic levels. Bio-functional assays, such as Transwell invasion, intracellular ATP, resazurin cell viability, fluorescence microscopy of filopodia formation, and antibody neutralization / cell rescue, were conducted in more NSCLC cell lines to validate changes identified from RNAseq and metabolomics analyses by confirming the corresponding EMT phenotypic changes.Results: RNAseq analysis shows that eATP significantly enriched expressions of genes involved in EMT temporarily, and similarly but non-identically to TGF-b after 2 and 6 hours of treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. Metabolomics analysis revealed eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar or identical to those induced by TGF-b. eATP-induced transcriptomic changes appeared smaller but earlier than TGF-b. Functional bioassays verified the RNAseq and metabolomics findings that eATP induced earlier and more invasion and formation of filopodia in A549 and H1299 cells, and restored viability of cancer cells treated with TGF-b-neutralizing antibodies. Conclusions: eATP-induced EMT, from gene expression changes and metabolic reprogramming, is similar but non-identical to that induced by TGF-b, and is independent of TGF-b. The smaller but earlier EMT-related changes induced by eATP, compared with TGF-b, could be largely explained by extracellular action of eATP and intracellular activities of macropinocytosis-internalized eATP. These strongly indicate that eATP is an emerging master inducer and regulator of EMT.