Extracellular and macropinocytosis internalized ATP work together to induce epithelial–mesenchymal transition and other early metastatic activities in lung cancer

Abstract

Background Extracellular ATP (eATP) was shown to induce epithelial–mesenchymal transition (EMT), a very important early process in metastasis, in cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer A549 and other cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis. Methods Floating cells, fencing, and transwell assays were used to show that ATP induces cell detachment, new colony formation, migration and invasion in human A549 and other lung cancer cells. Western blots were used to detect ATP-induced changes in EMT-related proteins; Confocal microscopy was used to demonstrate ATP-induced metastasis-related cell morphological changes. Inhibitors and siRNA knockdowns were used to determine P2X7’s involvement in the ATP-induced EMT. CRISPR–Cas9 knockout of the SNX5 gene was used to identify macropinocytosis’ roles in EMT and cancer cell growth both in vitro and in vivo. Student t-test and one-way ANOVA were used to determine statistical significance, P < 0.05 was considered significant. Results eATP potently induces expression of matrix metallopeptidases (MMPs), and detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-β and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis, slows down tumor growth, and changes tumor morphology in nude mice. Conclusions Collectively, these results show that eATP's functions in these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings shed light on eATP’s initiator and effector roles in almost every step in early metastasis, which calls for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identifies eATP and macropinocytosis as novel targets for potentially slowing down EMT and preventing metastasis.

Purinergic Signaling in the Hallmarks of Cancer

“…In the context of the Warburg effect, an interesting interpretation is that the metabolic role played by the internalized ATP serves as an energy supplement for the glycolytic ATP in cancerous cells. This phenomenon resulted only partially dependent on P2X7R [71].…”

“…The internalized ATP favored elevation of intracellular energy charge and promoted cancer growth, survival, and drug resistance, as well as the induction of EMT. More recently, these observations were extended to other neoplastic cell lines [71]. In the context of the Warburg effect, an interesting interpretation is that the metabolic role played by the internalized ATP serves as an energy supplement for the glycolytic ATP in cancerous cells.…”

“…In parallel, there was a reduction of the epithelial proteins E-cadherin and ZO-1. These results were ingeniously related with the exATP micropinocytosis process, since the genetic deletion of SNX5 (a gene involved with cell micropinocytosis) caused a significant reduction in cancer cell proliferation, migration and invasion [71].…”

“…At the same time, E-cadherin is down-regulated. These effects of P2X7R were mediated through PI3K/AKT phosphorylation and ERK1/2 signal transduction pathways [71,74].…”

Radiation Induced Metabolic Alterations Associate with Tumor Aggressiveness and Poor Outcome in Glioblastoma

“…Additionally, it can be internalized by tumor cells, increasing their intracellular ATP levels conferring metabolic reprogramming, increased tumor aggressivity, and treatment resistance (64)(65)(66)(67)(68). A recent lung cancer study has shown eATP to be involved in epithelial-to-mesenchymal transition, cell migration, and metastasis (69). While the biological functions of extracellular guanosine or eGTP are less studied than adenosine or eATP, their relative concentrations can covary, and biological functions of these nucleotides can cross-interact (70,71).…”