The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status
Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) s...
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2021-02-01
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Series: | International Journal of Molecular Sciences |
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Online Access: | https://www.mdpi.com/1422-0067/22/5/2369 |
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record_format |
Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Margarita Pustovalova Lina Alhaddad Taisia Blokhina Nadezhda Smetanina Anna Chigasova Roman Chuprov-Netochin Petr Eremin Ilmira Gilmutdinova Andreyan N. Osipov Sergey Leonov |
spellingShingle |
Margarita Pustovalova Lina Alhaddad Taisia Blokhina Nadezhda Smetanina Anna Chigasova Roman Chuprov-Netochin Petr Eremin Ilmira Gilmutdinova Andreyan N. Osipov Sergey Leonov The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status International Journal of Molecular Sciences non-small cell lung cancer cancer stem cells radioresistance Rad51 p53 epithelial-to-mesenchymal transition |
author_facet |
Margarita Pustovalova Lina Alhaddad Taisia Blokhina Nadezhda Smetanina Anna Chigasova Roman Chuprov-Netochin Petr Eremin Ilmira Gilmutdinova Andreyan N. Osipov Sergey Leonov |
author_sort |
Margarita Pustovalova |
title |
The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status |
title_short |
The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status |
title_full |
The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status |
title_fullStr |
The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status |
title_full_unstemmed |
The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status |
title_sort |
cd44high subpopulation of multifraction irradiation-surviving nsclc cells exhibits partial emt-program activation and dna damage response depending on their p53 status |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2021-02-01 |
description |
Ionizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines; 1D confined migratory behavior (wound healing); the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR; influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells’ radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential. |
topic |
non-small cell lung cancer cancer stem cells radioresistance Rad51 p53 epithelial-to-mesenchymal transition |
url |
https://www.mdpi.com/1422-0067/22/5/2369 |
work_keys_str_mv |
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doaj-dba9e2ac38c343c6b92f924e4d8d87672021-02-28T00:01:05ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01222369236910.3390/ijms22052369The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 StatusMargarita Pustovalova0Lina Alhaddad1Taisia Blokhina2Nadezhda Smetanina3Anna Chigasova4Roman Chuprov-Netochin5Petr Eremin6Ilmira Gilmutdinova7Andreyan N. Osipov8Sergey Leonov9School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaFSBI “National Medical Research Center for Rehabilitation and Balneology”, Ministry of Health of Russia, 121099 Moscow, RussiaFSBI “National Medical Research Center for Rehabilitation and Balneology”, Ministry of Health of Russia, 121099 Moscow, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaSchool of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, RussiaIonizing radiation (IR) is used for patients diagnosed with unresectable non-small cell lung cancer (NSCLC). However, radiotherapy remains largely palliative due to the survival of specific cell subpopulations. In the present study, the sublines of NSCLC cells, A549IR (p53wt) and H1299IR (p53null) survived multifraction X-ray radiation exposure (MFR) at a total dose of 60 Gy were investigated three weeks after the MFR course. We compared radiosensitivity (colony formation), expression of epithelial-mesenchymal transition (EMT) markers, migration activity, autophagy, and HR-dependent DNA double-strand break (DSB) repair in the bulk and entire CD44high/CD166high CSC-like populations of both parental and MFR survived NSCLC cells. We demonstrated that the p53 status affected: the pattern of expression of N-cadherin, E-cadherin, Vimentin, witnessing the appearance of EMT-like phenotype of MFR-surviving sublines; 1D confined migratory behavior (wound healing); the capability of an irradiated cell to continue to divide and form a colony of NSCLC cells before and after MFR; influencing the CD44/CD166 expression level in MFR-surviving NSCLC cells after additional single irradiation. Our data further emphasize the impact of p53 status on the decay of γH2AX foci and the associated efficacy of the DSB repair in NSCLC cells survived after MFR. We revealed that Rad51 protein might play a principal role in MFR-surviving of p53 null NSCLC cells promoting DNA DSB repair by homologous recombination (HR) pathway. The proportion of Rad51 + cells elevated in CD44high/CD166high population in MFR-surviving p53wt and p53null sublines and their parental cells. The p53wt ensures DNA-PK-mediated DSB repair for both parental and MFR-surviving cells irrespectively of a subsequent additional single irradiation. Whereas in the absence of p53, a dose-dependent increase of DNA-PK-mediated non-homologous end joining (NHEJ) occurred as an early post-irradiation response is more intensive in the CSC-like population MFR-surviving H1299IR, compared to their parental H1299 cells. Our study strictly observed a significantly higher content of LC3 + cells in the CD44high/CD166high populations of p53wt MFR-surviving cells, which enriched the CSC-like cells in contrast to their p53null counterparts. The additional 2 Gy and 5 Gy X-ray exposure leads to the dose-dependent increase in the proportion of LC3 + cells in CD44high/CD166high population of both parental p53wt and p53null, but not MFR-surviving NSCLC sublines. Our data indicated that autophagy is not necessarily associated with CSC-like cells’ radiosensitivity, emphasizing that careful assessment of other milestone processes (such as senescence and autophagy-p53-Zeb1 axis) of primary radiation responses may provide new potential targets modulated for therapeutic benefit through radiosensitizing cancer cells while rescuing normal tissue. Our findings also shed light on the intricate crosstalk between autophagy and the p53-related EMT, by which MFR-surviving cells might obtain an invasive phenotype and metastatic potential.https://www.mdpi.com/1422-0067/22/5/2369non-small cell lung cancercancer stem cellsradioresistanceRad51p53epithelial-to-mesenchymal transition |