Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis

Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis

Bibliographic Details
Main Author: Cao, Yanyang
Language:English
Published: Ohio University / OhioLINK 2019
Subjects:
Biology
Cellular Biology
Molecular Biology
Oncology
Tumor microenvironment
Invasion
EMT
ATP internalization
TGF-beta
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ohiou156984033112675
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ohiou1569840331126752021-08-03T07:12:54Z Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis Cao, Yanyang Biology Cellular Biology Molecular Biology Oncology Tumor microenvironment Invasion EMT ATP internalization TGF-beta Intratumoral extracellular ATP (eATP), at levels of 100-700 μM or 10<sup>3</sup> to 10<sup>4</sup> times higher than in normal tissues, has been known to induce epithelial-mesenchymal-transition (EMT) of 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 lung 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. Floating cells, fence assay, 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. Inhibitor 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’s t-test and one-way ANOVA were used to determine statistical significance, P<0.05 was considered significant. Here we report that eATP, at the concentrations reported in tumors, potently induces expression of matrix metallopeptidases (MMPs), detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-beta 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 the macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis, slows down tumor growth, and changes tumor morphology in nude mice. Collectively, these results show that eATP functions on these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings which shed light on eATP’s initiator and effector roles in almost every step in early metastasis call for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identify eATP and macropinocytosis as novel targets for potentially slowing down EMT, preventing metastasis, and reducing metastasis-related death in cancer patients. 2019 English text Ohio University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ohiou156984033112675 http://rave.ohiolink.edu/etdc/view?acc_num=ohiou156984033112675 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
collection NDLTD
language English
sources NDLTD
topic Biology
Cellular Biology
Molecular Biology
Oncology
Tumor microenvironment
Invasion
EMT
ATP internalization
TGF-beta
spellingShingle Biology
Cellular Biology
Molecular Biology
Oncology
Tumor microenvironment
Invasion
EMT
ATP internalization
TGF-beta
Cao, Yanyang
Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
author Cao, Yanyang
author_facet Cao, Yanyang
author_sort Cao, Yanyang
title Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
title_short Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
title_full Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
title_fullStr Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
title_full_unstemmed Roles of Extracellular ATP in Induction of Epithelial-Mesenchymal Transition and Other Early Steps of Metastasis
title_sort roles of extracellular atp in induction of epithelial-mesenchymal transition and other early steps of metastasis
publisher Ohio University / OhioLINK
publishDate 2019
url http://rave.ohiolink.edu/etdc/view?acc_num=ohiou156984033112675
work_keys_str_mv AT caoyanyang rolesofextracellularatpininductionofepithelialmesenchymaltransitionandotherearlystepsofmetastasis
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