Extracellular Atp Is Internalized Through Not Only Macropinocytosis But Also Other Endocytic Process And Promotes Growth Of Human Lung Cancer Cells With Ras Mutations

Within a tumor, extracellular ATP (exATP) concentrations are in the range of several hundred micromolars or higher, 103-104 times higher than those in normal tissues1. We recently showed that extracellular ATP, within the intratumoral concentrations, was internalized by Ras-mutated (Rasmut) A549 human lung cancer cells through macropinocytosis, elevated intracellular ATP levels and rescued cancer cells under various stresses2. However, macropinocytosis3 alone could not account for all the elevated intracellular ATP increase. Here, we show that other endocytic processes, in addition to macropinocytosis, also contributed to the internalization of exATP in A549 and H1299 human lung cancer cells. Cell viability assays, intracellular ATP luminescence measurement assays, fluorescent microscopy were used in this study. First, high-molecular-weight (HMW) dextran uptake assay indicated that A549 and H1299, both Rasmut cancer cells, exhibited a phenotype of macropinocytosis. The colocalization of the red fluorescent HMW dextran and green nonhydrolyzable ATP analog inside the cells indicated that extracellular ATP was internalized by macropinocytosis. In contrast, significantly fewer macropinosomes were found in NL-20 noncancerous lung cells. In addition, the colocalization of low-molecular-weight (LMW) dextran, an endocytic tracer, and ATP analog indicated the internalization of exATP through non-macropinocytic-endocytosis in both lung cancer cell lines. Interestingly, when both red HMW dextran and green LMW dextran were applied to cancer cells, not only colocalization, but also some green-only endosomes were observed, strongly suggesting that both endocytic processes were responsible for exATP internalization. Collectively, these data indicated for the first time that other endocytic processes in addition to macropinocytosis contributed to the internalization of exATP in human lung cancer cells with Rasmut. This finding may significantly enhance our understanding of energy hemostasis within cancer cells and the Warburg effect, which in turn facilitates the development of anticancer therapeutics targeting the endocytic processes and Ras mutations as well as novel anticancer strategies. 1. Pellegatti, et al. (2008). PLoS ONE 3, e2599. 2. Qian, et al. (2014). Cancer Letters 351, 242-251. 3. Commisso, et al. (2013). Nature 497, 633-637. Citation Format: Yanrong Qian, Xuan Wang, Yunsheng Li, Yanyang Cao, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP is internalized through not only macropinocytosis but also other endocytic process and promotes growth of human lung cancer cells with Ras mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2015-1173