CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status

Abstract

Cancer stem cells (CSCs) play a critical role in the initiation, progression and therapy relapse of many cancers including non-small cell lung cancer (NSCLC). Here, we aimed to address the question of whether the FACS-sorted CSC-like (CD44+ &CD133+) vs. non-CSC (CD44−/CD133− isogenic subpopulations of p53wt A549 and p53null H1299 cells differ in terms of DNA-damage signaling and the appearance of “dormant” features, including polyploidy, which are early markers (predictors) of their sensitivity to genotoxic stress. X-ray irradiation (IR) at 5 Gy provoked significantly higher levels of the ATR-Chk1/Chk2-pathway activity in CD44−/CD133− and CD133+ subpopulations of H1299 cells compared to the respective subpopulations of A549 cells, which only excited ATR-Chk2 activation as demonstrated by the Multiplex DNA-Damage/Genotoxicity profiling. The CD44+ subpopulations did not demonstrate IR-induced activation of ATR, while significantly augmenting only Chk2 and Chk1/2 in the A549- and H1299-derived cells, respectively. Compared to the A549 cells, all the subpopulations of H1299 cells established an increased IR-induced expression of the γH2AX DNA-repair protein. The CD44−/CD133− and CD133+ subpopulations of the A549 cells revealed IR-induced activation of ATR-p53-p21 cell dormancy signaling-mediated pathway, while none of the CD44+ subpopulations of either cell line possessed any signs of such activity. Our data indicated, for the first time, the transcription factor MITF–FAM3C axis operative in p53-deficient H1299 cells, specifically their CD44+ and CD133+ populations, in response to IR, which warrants further investigation. The p21-mediated quiescence is likely the predominant surviving pathway in CD44−/CD133− and CD133+ populations of A549 cells as indicated by single-cell high-content imaging and analysis of Ki67- and EdU-coupled fluorescence after IR stress. SA-beta-galhistology revealed that cellular-stress-induced premature senescence (SIPS) likely has a significant influence on the temporary dormant state of H1299 cells. For the first time, we demonstrated polyploid giant and/or multinucleated cancer-cell (PGCC/MGCC) fractions mainly featuring the progressively augmenting Ki67low phenotype in CD44+ and CD133+ A549 cells at 24–48 h after IR. In contrast, the Ki67high phenotype enrichment in the same fractions of all the sorted H1299 cells suggested an increase in their cycling/heterochromatin reorganization activity after IR stress. Our results proposed that entering the “quiescence” state rather than p21-mediated SIPS may play a significant role in the survival of p53wt CSC-like NSCLC cells after IR. The results obtained are important for the selection of therapeutic schemes for the treatment of patients with NSCLC, depending on the functioning of the p53 system in tumor cells.

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status

“…In the present study, the degree of MGCC enrichment was highest in LN-229 cells, compared to the modest (but still significant) enrichment in U-87 and the marginal one in U-251 cells ( Figure 1 d–f), suggesting that transactivation is functional either in wild-type or mutated p53 and its driven pathway might be associated with MGCC formation efficacy. This observation brings more support to our preceding hypothesis that under genotoxic and nutrient stress, such as IR exposure and serum starvation, functional p53 may promote entering a safer, presumably “dormant” state to preserve solid tumor cells from death irrespectively of their stemness [ 92 ].…”

“…In this regard, we did not investigate polyploidy and stemness acquisition in our present study. Notwithstanding, such an observed early stress-induced response correlates quite well with time of appearance of “dormancy-related” features, including polyploidy, in CSC-like (CD44+/CD133+) vs. non-CSC (CD44-/CD133-) isogenic subpopulations of A549 and H1299 non-small lung cancer cells (NSCLCs) as an early marker (predictor) of their behavior after the same type of genotoxic stress [ 92 ]. Moreover, the behavior and dynamics of each feature’s appearance in lymphoblast [ 12 , 91 ] and solid (NSCLCs or GBM studied by us) tumors are supposed to be different.…”

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons

“…However, we could not detect an increased expression of the stemness transcription factors SOX2, NANOG, and POU5F1 in the CD44 + populations. This is in contrast to other authors, who found an increased expression in CD44 + NSCLC cells compared to CD44 − cells [17,37]. However, they did not grow their cells as tumorspheres, prior to sorting, and the expression of pluripotency factors was much higher than adherent cells since tumorsphere culture is an enrichment of CSCs [38].…”

Dormant cancer cells and polyploid giant cancer cells: The roots of cancer recurrence and metastasis

“…124,126,146 P53 drives cells into a safer and more favourable dormant state, enhancing the adaptability and survival of PGCCs to irradiation stress. 66,147 The CHK1, CHK2-pCDC25C-Ser216cyclin B1-CDK1 and AURKA-Polo-like kinase 1 (PLK1)-pCDC25C-Ser198-cyclin B1-CDK1 signalling pathways are participant of PGCCs formation. 25,126 ataxia telangiectasiamutated gene (ATM)/ataxia telangiectasia mutated and Rad3-related (ATR)-P53-p21 axis induces cell dormancy and cell cycle arrest.…”