Several unique features of eATP and macropinocytosed eATP support them as being a potential master inducer and regulator of EMT. First, ATP is an extracellular messenger for activating purinergic receptor (PR) mediated signaling, which has been implicated in EMT [44–46], providing the basis for specificity for ATP-triggered signaling and actions. ATP is a protein phosphorylation donor participating in almost all intracellular signaling pathways. In PR signaling, eATP not only induces PR activation, but also enhances PR signaling by providing extra phosphate donors when eATP is internalized to elevate intracellular ATP concentrations. Second, ATP is also a versatile transcription cofactor, participating in a wide variety of transcriptional activities such as DNA unwinding, transcription initiation, elongation, and termination [30–34]. Furthermore, ATP is also a cofactor in energy-required enzymatic reactions, accelerating reaction rates. These properties make ATP stand out as a potential inducer and regulator for EMT, a process that relies on well-regulated changes in signal transduction, gene expression (transcription), translation, and metabolism. More directly related to cell motility and metastasis, ATP is a well-known danger signal for bacteria and cancer cells [47, 48]. Elevated ATP concentrations in the environment functions as a warning signal to bacteria and animal cells for the incoming danger and telling them to flee for a safer environment. Apparently, this conserved activity is hijacked and utilized by cancer cells to signal imminent danger of their original sites within tumors when the conditions in TME is deteriorating due to hypoxia and shortage of nutritional supply. It is conceivable that eATP-induced EMT is one of the mechanisms utilized by cancer cells in tumors preparing cancer cell and enabling them for departure, reseeding in the same tumor, invasion and even metastasis. Some major remaining questions include: how much, at what levels, at what cellular locations, and in what relationship to TGF-b, eATP participates in EMT? Our current study was one of the first steps towards answering these key questions for better understanding of the EMT induction process by using a combinatorial study strategy of RNAseq, metabolomics, and functional assays.
The Venn graphs of the RNAseq analysis show that, like TGF-b, eATP upregulated many genes, and downregulated many other genes. Most of these genes are upregulated or downregulated similarly by eATP and TGF-b (Table 1), strongly suggesting that eATP induced and regulated the same process as TGF-b, namely EMT. These changes also indicate that the eATP treatment did not randomly induce gene expressions but only those related to and needed for EMT. Secondly, also like TGF-b, eATP exhibited time-dependent gene expressions. The 6-hour treatment led to an appearance of the expression of some new genes that did not show up at the 2-hour treatment and a disappearance of the expression and downregulation of some other genes. These expression pattern changes suggest that eATP not only induces EMT, but also temporally regulates EMT, orchestrating the progress by expressing the right genes at the right times and at right levels.
On the first look, eATP induced fewer significant changes in upregulated and downregulated genes compared with TGF-β. A closer examination of the RNAseq data reveals that eATP induced changes of expression in about as many genes as TGF-β, but just not to the level of statistical significance determined by the RNAseq software (unpublished observations). Similar phenomenon was also observed in metabolomics data (Fig. 4e and 5d). The multi-functionality and multi-locality of ATP and internalized eATP might be behind the apparent differences. It is possible that the elevated iATP concentrations (Fig. 5d) drive the accelerated signal transduction and biochemical reactions while maintaining the expression of the enzyme genes and metabolites involved in these reactions at levels lower than those found in TGF-β-treated cells (Table 1, and Fig. 4e and 5d).
For some of those M-type genes significantly altered by both eATP and TGF-b, eATP-induced genes tend to show higher Log2fc values at 2 hr than those induced by TGF-b, while genes induced by TGF-b tend to have higher Log2fc values at 6 hr than eATP-induced genes (Table 1). This pattern of gene expression suggests that eATP induces gene expression changes earlier and possibly faster than TGF-b, consistent with the observation that eATP induced faster morphological changes than TGF-b (Fig. 3 and [29]). This is also consistent with the notion of the multi-functionality and multi-locality of eATP.
Unlike the RNAseq profiles at 2 and 6 hours after the inductions, which reflected the changes in early and late stages of gene expression during EMT, the metabolomics profile represents changes at the metabolic and therefore phenotypic levels associated with EMT [44, 45]. The metabolomics data provides evidence, in addition to the RNAseq gene expression data, that eATP induced a gene expression and metabolic profile similar to that induced by TGF-b (Fig. 2). The specific altered pathways and metabolite levels, compared with TGF-b, are clear indications that the metabolic state induced by eATP is similar to that induced by TGF-b, and is indeed a state corresponding to EMT.
These changes indicate that eATP induced pattern alterations from transcription to metabolic levels with characteristics expected for an induced EMT state, which is also similar to EMT state induced by TGF-b. These findings have never been reported before.
We recently reported the observation that eATP induced both migration and invasion [29]. Our current invasion assays further expand the study by showing dose-dependent comparison between eATP and TGF-b in not only A549 cells, but also in a second lung cancer cell line H1299 (Fig. 3a and 3b). The new result shows that this is not a single cell line phenomenon but a potentially prevalent activity of eATP among cancer cells. The doses of eATP used in this study were the same as the concentration range of eATP found in TME [16–19], implying its roles in vivo. Our subsequent fluorescent microscopy study revealed that the eATP treatment led to an earlier formation of filopodia-like protrusions in A549 and H1299 cells in a time-dependent manner (Fig. 4a and 4b) than TGF-b. This result provides a first piece of visual evidence for earlier EMT-related morphological changes induced by eATP.
Eleven genes, which were significantly upregulated and completely conserved in eATP and TGF-b treated cells at both 2 and 6 hours, were identified (Table 1). Several genes in this group, including Sox8, BMP6, MMP10, and IL-1A, are known to play roles in EMT. other genes including STC1, BLOC1S6, are not known to be involved in EMT. It is particularly interesting to find that ATP6V1G2, an untranslated long transcript fused between an ATPase gene and an RNA splicing gene, is also included in this group. The presence and conservation of ATP6V1G2 long RNA suggest the potential regulatory functions of the long transcript. The presence of the transcript in TGF-b treated cells also suggests that this is not an eATP-unique phenomenon. It is likely to be more fundamental for induction and regulation of EMT. It is also noteworthy that 8 out of the 11 (~ 73%) genes were induced to the higher level by eATP than TGF-b at 2 hours, while only 4 out 11 genes (~ 36%) were induced to the higher level by eATP than TGF-b at 6 hours. This provides another piece of evidence that eATP induces EMT earlier than TGF-b in addition to the invasion assay and then filopodia formation assay.
The study for the functional relationship revealed that eATP and TGF-b do not show an additive effect on invasion at higher (TME) concentrations (Fig. 5a and 5b), suggestive of the overlapping of the invasion-inducing activities of the two molecules. When pan-TGF-b neutralizing antibodies were exogenously added, diminished invasion was observed. The decrease was reversed, restored, and the viability was even enhanced by the addition of eATP (Fig. 5c and 5d). These results assert that ATP can not only induce invasion without the addition of TGF- b, but also in the absence of TGF- b induced signaling, further supporting the notion that eATP functions independent of TGF- b in the induction of EMT.
The faster EMT induction by eATP than TGF-b might be related to macropinocytosis-mediated ATP internalization, which results in a large elevation of intracellular ATP (iATP) levels. The ATP assay confirmed this speculation in that eATP induced large dose-dependent iATP elevations in both A549 and H1299 cells, while TGF-b did not (Fig. 6a & 6b). It is conceivable that the highly elevated iATP enhanced protein phosphorylation in signal transduction, accelerated biochemical reactions and cell morphology changes, and increased cell motility. We previously demonstrated that some of these activities were blocked when macropinocytosis, a primary hallmark of cancer metabolism [49], was inhibited [29]. A549 and H1299 cells are known to exhibit macropinocytosis [50, 51]. In addition, iATP directly participates in induction and regulation of gene expressions in cancer cells as a transcriptional cofactor. All these combined together may account for the greater invasion rates compared with TGF-b (Fig. 3a & 3b). Many other cancer cell lines of other cancer types also show macropinocytosis [20–22]. Although macropinocytosis is an energy-consuming (ATP-consuming) process, our study implies that cancer cells use it to obtain sufficient “free” ATP from the TME to drive and sustain macropinocytosis and the biochemical reactions and protein phosphorylation without using as much endogenously synthesized ATP.
Based on all the previous studies related to ATP-induced EMT [14, 29] and this study, here we propose a significantly updated hypothetical model for how eATP induces and regulates EMT spatially and temporally in human lung cancers (Fig. 7e). First, eATP, at the concentration range found in TME [16–19], functions extracellularly by binding and activating various purinergic receptors (PR) located on plasma membrane, leading to PR-mediated specific signaling for EMT induction [39, 52]. Exactly which PR(s) are activated depend on the specific eATP concentration as different PRs have different affinities for ATP. This part of eATP activity is similar or identical to the mechanism of TGF-b mediated PR signaling as TGF-b induces ATP exocytosis and subsequent ATP-PR binding/activating (13–14) with the exception that eATP levels in the TME may be higher than the eATP level achieved by TGF-b -mediated ATP exocytosis. This is because eATP in the TME is from multiple sources [53, 54] in addition to TGF-b-induced exocytosis [13, 14]. Simultaneously with the PR signaling, eATP is also internalized by macropinocytosis [20–22, 25–27], greatly enhancing the level of intracellular ATP (iATP) by at least 30–50% within 2–3 hours [25–27]. The elevated iATP, in turn, accelerates all biochemical / enzymatic reactions inside the cell partly driven by ATP, including both ATP hydrolysis in metabolic reactions and protein phosphorylation in signal transduction. Furthermore, ATP is versatile transcriptional cofactor, directly participating in and augmenting gene expression by ways of double strand DNA unwinding, transcription initiation, elongation, and other steps in transcription [30–34]. All these processes working concurrently at different subcellular locations and at various levels of biological function result in induction and spatial and temporary regulation of EMT and steps beyond EMT in metastasis. While the specificity of the gene expression induced by eATP is likely to be originated from the PR signaling, the intensity of the gene expression is likely to be regulated by the other intracellular functions of eATP and potential negative feedback loops between gene transcription rates and enhanced enzymatic activities induced by augmented protein phosphorylation and/or faster enzymatic reactions (and therefore altered metabolite levels) driven by higher iATP levels. Thus, this model not only explains how eATP induces TGF-b-like EMT, but also explains why eATP induces EMT somewhat differently from TGF-b-induced EMT at the levels of transcription and biochemical reactions, resulting in earlier morphological / functional changes. Additional studies are needed for the final validation of this hypothetical model.