Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma
Background: Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarc...
Main Authors: | , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
SAGE Publishing
2020-09-01
|
Series: | Therapeutic Advances in Medical Oncology |
Online Access: | https://doi.org/10.1177/1758835920956900 |
id |
doaj-2d822bbd82db4cf9909685368289b740 |
---|---|
record_format |
Article |
spelling |
doaj-2d822bbd82db4cf9909685368289b7402020-11-25T03:14:04ZengSAGE PublishingTherapeutic Advances in Medical Oncology1758-83592020-09-011210.1177/1758835920956900Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcomaXiaoyang LiDylan C. DeanGregory M. CoteLee ZouFrancis J. HornicekShengji YuZhenfeng DuanBackground: Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target. Methods: The osteosarcoma tissue microarrays constructed from 70 patient specimens underwent immunohistochemistry to quantify ATR and activated phospho-ATR (pATR) expression and their correlation with clinical outcomes. ATR sublocalization within the metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis, and migration were evaluated following treatment with ATR siRNA or the selective inhibitor Berzosertib. Antitumor effects were determined with ex vivo three-dimensional (3D) culture models, and the impacts on the DNA damage repair pathways were measured with Western blotting. Results: Elevated ATR and activated pATR expression correlated with shorter patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a mechanism related to DNA replication. ATR knockdown with siRNA or inhibition with Berzosertib suppressed cell proliferation in a time- and dose-dependent manner and induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increasing γH 2 AX expression and PARP cleavage, consistent with the interference of DNA damage repair. The ATR inhibitor Berzosertib also produced the characteristic cytoplasmic vacuolization preceding cell death, and suppressed ex vivo 3D spheroid formation and cell motility. Conclusion: The faithful dependence of cells on ATR signaling for survival and progression makes it an emerging therapeutic target in metastatic osteosarcoma.https://doi.org/10.1177/1758835920956900 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xiaoyang Li Dylan C. Dean Gregory M. Cote Lee Zou Francis J. Hornicek Shengji Yu Zhenfeng Duan |
spellingShingle |
Xiaoyang Li Dylan C. Dean Gregory M. Cote Lee Zou Francis J. Hornicek Shengji Yu Zhenfeng Duan Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma Therapeutic Advances in Medical Oncology |
author_facet |
Xiaoyang Li Dylan C. Dean Gregory M. Cote Lee Zou Francis J. Hornicek Shengji Yu Zhenfeng Duan |
author_sort |
Xiaoyang Li |
title |
Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
title_short |
Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
title_full |
Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
title_fullStr |
Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
title_full_unstemmed |
Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
title_sort |
inhibition of atr-chk1 signaling blocks dna double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma |
publisher |
SAGE Publishing |
series |
Therapeutic Advances in Medical Oncology |
issn |
1758-8359 |
publishDate |
2020-09-01 |
description |
Background: Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target. Methods: The osteosarcoma tissue microarrays constructed from 70 patient specimens underwent immunohistochemistry to quantify ATR and activated phospho-ATR (pATR) expression and their correlation with clinical outcomes. ATR sublocalization within the metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis, and migration were evaluated following treatment with ATR siRNA or the selective inhibitor Berzosertib. Antitumor effects were determined with ex vivo three-dimensional (3D) culture models, and the impacts on the DNA damage repair pathways were measured with Western blotting. Results: Elevated ATR and activated pATR expression correlated with shorter patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a mechanism related to DNA replication. ATR knockdown with siRNA or inhibition with Berzosertib suppressed cell proliferation in a time- and dose-dependent manner and induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increasing γH 2 AX expression and PARP cleavage, consistent with the interference of DNA damage repair. The ATR inhibitor Berzosertib also produced the characteristic cytoplasmic vacuolization preceding cell death, and suppressed ex vivo 3D spheroid formation and cell motility. Conclusion: The faithful dependence of cells on ATR signaling for survival and progression makes it an emerging therapeutic target in metastatic osteosarcoma. |
url |
https://doi.org/10.1177/1758835920956900 |
work_keys_str_mv |
AT xiaoyangli inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT dylancdean inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT gregorymcote inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT leezou inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT francisjhornicek inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT shengjiyu inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma AT zhenfengduan inhibitionofatrchk1signalingblocksdnadoublestrandbreakrepairandinducescytoplasmicvacuolizationinmetastaticosteosarcoma |
_version_ |
1724644744101888000 |