The CD44high Subpopulation of Multifraction Irradiation-Surviving NSCLC Cells Exhibits Partial EMT-Program Activation and DNA Damage Response Depending on Their p53 Status

Abstract

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.

Opposing Roles of Wild-type and Mutant p53 in the Process of Epithelial to Mesenchymal Transition

“…The effect of p53 status on EMT was shown for several carcinomas, including bladder ( Wang et al, 2016 ) and lung cancers ( Pustovalova et al, 2021 ). Also, the prognostic value of p53 and Snail status was revealed on the biopsy samples of endometrial carcinomas ( Dragomirescu et al, 2018 ).…”

GATA3 Exerts Distinct Transcriptional Functions to Regulate Radiation Resistance in A549 and H1299 Cells

“…The radioresistance of p53null H1299 cells results in cell cycle checkpoint disturbance and a higher tetraploid ratio compared to that of p53wt A549 cells. It has been demonstrated in the literature that two types of lung adenocarcinoma cells (A549 and H1299) have different sensitivities to radiation, and after conventionally fractionated irradiation regimens, the two types of cells exhibit different apoptosis, metabolic activity, and EMT transformation [ 36 ]. This fully demonstrates that nonsmall cell lung cancer (NSCLC) treatments should become more personalized according to the status of key protein molecules in tumor tissue [ 34 ].…”

Curcuminoids as Modulators of EMT in Invasive Cancers: A Review of Molecular Targets With the Contribution of Malignant Mesothelioma Studies

“…A first report described the importance of a chaperone protein of histone H3-H4, the anti-silencing function 1 (ASF1B), which promotes cell proliferation, migration, and invasion through the modulation of the p53mediated EMT signaling pathway (Wang et al, 2022). Secondly, Pustovalova et al shed light on the crosstalk between autophagy and this pathway (Pustovalova et al, 2021). Thirdly, Avşar Abdik (2021) demonstrated how the p53 status affected mature adipocyte-mediated proliferation, emphasizing the importance of targeting the tumor microenvironment.…”