Abstract 2019: Extracellular ATP induces EMT and metastatic activities in non-small cell lung cancer cells

Abstract

Abstract Cancer is a leading cause of death worldwide. Metastasis, the spread of cancer cells from the primary tumors to distant organs, is responsible for approximately 90% of all cancer-related deaths. Metastasis is a multistep process in which loss of cell-cell adhesion, increased proteolysis, and cell motility have been shown to be critical steps. Recent studies have indicated that intratumoral ATP concentration is ~1,000 times higher than normal tissues of the same cell origins in the range of at least several hundred μM. Extracellular ATP (eATP), a tumor microenvironment molecule, potentially has a crucial role in supporting tumor invasion and metastasis. Our previous studies showed that eATP is internalized by cancer cells through macropinocytosis and other endocytosis and promotes cancer cell growth, survival, and drug resistance (1-4). Based on all these, we hypothesize that eATP functions as both a signaling molecule and an energy source to stimulate epithelial mesenchymal transition (EMT) and metastasis. Various bioassays were used in human non-small cell lung cancer (NSCLC) A549 and HOP-92 cells to test our hypothesis. Our results reveal that eATP treatment led to a substantially increased number of floating cancer cells, and these cells were viable and formed clones in clonogenic assays. Fence assay indicated that eATP not only induces cancer cell detachment but also increases the number of secondary colonies formed. Moreover, treatment of eATP induces a dose- and time-dependent increase in the migration and invasive capacities of the A549 and HOP-92 cells. Confocal images demonstrated that eATP induces the formation of F-actin-dependent filopodia necessary for cell migration. Using Western blot analysis, we found that the expression of epithelial markers was significantly reduced, while the mesenchymal markers were increased after eATP treatment. Purinergic receptor (PR) inhibitors and P2X7 receptor knockdown only slightly attenuated these effects, suggesting PR signaling is only partially responsible for eATP-induced migration and invasion. These results indicate eATP is an EMT and metastasis stimulator and imply novel targets for inhibiting/preventing metastasis.