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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?

SUBMITTER: Cao Y

PROVIDER: S-EPMC6771108 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Cao Yanyang Y Wang Xuan X Li Yunsheng Y Evers Maria M Zhang Haiyun H Chen Xiaozhuo X

Cancer cell international 20191001

<h4>Background</h4>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 ...[more]

PMID: 31582910

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Project description:Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.

Project description:Inflammation driven by intracellular activation of the NLRP3 inflammasome is involved in the pathogenesis of a variety of diseases including vascular pathologies. Inflammasome specks are released into the extracellular compartment from disrupting pyroptotic cells. The potential uptake and function of extracellular NLRP3 inflammasomes in human coronary artery smooth muscle cells (HCASMC) are unknown. Fluorescently labeled NLRP3 inflammasome particles were isolated from a mutant NLRP3-YFP cell line and used to treat primary HCASMC for 4 and 24 h. Fluorescent and expressional analyses showed that extracellular NLRP3-YFP particles are internalized into HCASMC, where they remain active and stimulate intracellular caspase-1 (1.9-fold) and IL-1β (1.5-fold) activation without inducing pyroptotic cell death. Transcriptomic analysis revealed increased expression level of pro-inflammatory adhesion molecules (ICAM1, CADM1), NLRP3 and genes involved in cytoskleleton organization. The NLRP3-YFP particle-induced gene expression was not dependent on NLRP3 and caspase-1 activation. Instead, the effects were partly abrogated by blocking NFκB activation. Genes, upregulated by extracellular NLRP3 were validated in human carotid artery atheromatous plaques. Extracellular NLRP3-YFP inflammasome particles promoted the secretion of pro-atherogenic and inflammatory cytokines such as CCL2/MCP1, CXCL1 and IL-17E, and increased HCASMC migration (1.8-fold) and extracellular matrix production, such as fibronectin (5.8-fold) which was dependent on NFκB and NLRP3 activation. Extracellular NLRP3 inflammasome particles are internalized into human coronary artery smooth muscle cells where they induce pro-inflammatory and pro-atherogenic effects representing a novel mechanism of cell-cell communication and perpetuation of inflammation in atherosclerosis. Therefore, extracellular NLRP3 inflammasomes may be useful to improve the diagnosis of inflammatory diseases and the development of novel anti-inflammatory therapeutic strategies.

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

Publications

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

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