ΔNp63α inhibits the radiation-induced DDR through transcriptional repression

To investigate the function of ΔNp63α as a p53 repressor in the radiation response, we first performed siRNA knockdown experiments in HMECs expressing the mammary basal cell markers cytokeratin (CK) 5/14 and integrin α6 (CD49f). HMECs were confirmed by FCM analysis to be a single population and expressed ΔNp63α (72 kDa) almost exclusively among all p63 isotypes in the nucleus (Fig. 1a-d and Fig. S1a and b). After p63-siRNA (sip63) treatment, ΔNp63α mRNA and protein were reduced to 10–20% of the levels in scr-treated cells at 24 h, but at 96 h, they had recovered to the level observed before sip63 treatment (Fig. 1c and d and Fig. S1c). At this time, the expression of genes upregulated by ΔNp63α, such as CK14 [31] and Fst [14], was decreased (Figs. 1c and 2b and Fig. S1e).

Fig. 1

ΔNp63α knockdown experiments with HMECs. (a) Domain structure of human p53 and p63 isotypes. TAp63 and ΔNp63, highlighting the transactivation (TA), DNA-binding, oligomerization, and SAM domains. (b) mRNA expression concordant with positions (1)-(4) shown in Fig. 1a. hiPSCs were used as a negative control for p63. (c) Time-dependent variation in ΔNp63α and cytokeratin 14 (CK14) protein expression in p63 siRNA (sip63)- or scramble siRNA (scr)-treated HMECs. The arrow indicates the ΔNp63α protein band. (d) Representative immunofluorescence (IF) images of HMECs treated with sip63 and stained with ΔNp63 and CK14 antibodies. Red and green indicate ΔNp63 and CK14, respectively. Scale bar, 10 μm. (e, f) Time-dependent variations in ΔNp63 mRNA (e) and ΔNp63α protein (f) contained in whole-cell extracts of HMECs treated with sip63 for 24 h after irradiation. (g) Measurement of DNA damage response (DDR)-marker mRNA expression in sip63-treated HMECs. (h) Western blotting analyses of BAX and p21 proteins. (i) Comparison of EdU-positive frequencies in sip63- and scr-treated cells, which was evaluated by flow cytometry (FCM). (j) Frequencies of apoptotic cells detected by FCM in sip63- or scr-treated HMECs. All values in mRNA expression data were scaled to the expression level of GAPDH as an internal control. Data represent the means and SEs of at least three independent assays. *P < 0.05, **P < 0.01 by Student’s t test

Fig. 2

RNA transcriptome analysis of HMECs. (a) Left: K-means clustering heatmaps showing gene expression in siRNA-treated HMECs at 24 h post-irradiation. Right: Pathway enrichment analysis of scr- and sip63-treated groups in each cluster based on the Gene Ontology (GO) biological process database. Clusters A and B contain 295 and 205 genes, where upregulated and downregulated genes are depicted in red and blue, respectively. (b) Biclustering analysis based on the BCCC method for siRNA-treated HMECs at 24 h post-irradiation

The sip63 sequence used in this study targets the DNA-binding domain of p63 (NM_001114980.2: position 714–732) and is not homologous to any other gene, as confirmed by NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and GGGenome (https://gggenome.dbcls.jp/). RNA-seq and RT–qPCR results showed that TP53 mRNA expression was increased after sip63 treatment (Fig. 2b and Figs. S1d and S3). Since the objective in this study was to examine the p53 repressor activity and functionality of ΔNp63α in radiation-induced DDR, we chose to irradiate the cells between 30 and 42 h after sip63 treatment when ΔNp63α was sufficiently attenuated and p53 expression was constant (Fig. S1d). The ΔNp63α expression level remained almost unchanged within 24 h after irradiation (Fig. 1e and f), while the p53 expression level showed time-dependent enhancement (Fig. 1f). On the other hand, the amount of p53 detected in the scr-treated group was more abundant than that in the sip63-treated group. In the study of radiation-induced breast cancer, experimental animals, such as rats, are exposed to 4 Gy of γ (X) irradiation to induce tumour formation [32]. Therefore, the dose used in this experiment was set at 4 Gy.

Radiation-induced DDR in sip63-treated cells was investigated using RT–qPCR and RNA-seq. RT–qPCR quantified the expression levels of the apoptosis-related genes BAX and NOXA and the cell cycle arrest-related genes CDKN1A and CDKN2A, which are regulatory targets of the p53 protein (Fig. 1g). After irradiation, p53 is phosphorylated by kinases such as ATM to escape ubiquitin degradation and binds to the cis-elements of these genes to activate transcription [2, 3]. In the scr-treated group, the expression level of each gene increased slightly within 24 h after irradiation (no significant difference). In contrast, in the sip63-treated group, these gene expression levels showed a significant time-dependent increase (Fig. 1g). In particular, the responses of BAX and CDKN1A were remarkable, and there was a significant difference in BAX between the scr-treated and sip63-treated groups even in the absence of irradiation (Fig. 1g, P0Gy − 0 Gy<0.05). To confirm this result, we examined the temporal changes in BAX and p21 after irradiation by Western blot analysis (Fig. 1h). The results showed that the overall expression of both proteins was higher in the sip63-treated group, which was consistent with the results of RT–qPCR analysis.

To further confirm these results, we measured the EdU uptake rate and radiation-induced apoptotic cells by FCM analysis (Fig. 1i and j). After sip63 treatment, the EdU uptake rate showed a decrease from approximately 15–7%, while the percentage of cells in G0/G1 phase increased to approximately 90% (Fig. 1i and Fig. S2a). After irradiation to the sip63-treated group, the EdU uptake rate showed a significant decrease from 7.4 to 5.9% (P < 0.05, Fig. 1i). The detection of apoptotic CC3-positive cells after irradiation showed that the percentage of apoptotic cells increased in a time-dependent manner within 24 h post-irradiation, ranging from 2% to approximately 10% (Fig. 1j and Fig. S2b). These results are consistent with the results of mRNA and protein analyses (Fig. 1g and h). In the scr-treated group, the proportion in S phase was also decreased at 24 h post-irradiation, and the G0/G1 phase population became dominant (Fig. S2a). The p21 protein is a strong CDK inhibitor and inhibits the G1-S phase transition [2, 3]. Therefore, this result is consistent with the results of the CDKN1A mRNA and p21 protein analyses (Fig. 1g and h). The cell viability in the sip63-treated group at 48 h post-irradiation was 67%, which was approximately 10% lower than that in the scr-treated group (Fig. S1f). This result may also indicate an increase in radiation-induced apoptotic cells in the sip63-treated group.

To validate the results of the siRNA-based knockdown experiments, we also performed ΔNp63α knockdown experiments using the CRL4CRBN-thalidomide system (Fig. S1g). Thalidomide is a celebron (CRBN) modulator that has been reported to bind to the E3 ubiquitin ligase substrate receptor, thereby enhancing the binding of CRBN to the neosubstrate ΔNp63α and contributing to the ubiquitination of ΔNp63α [33]. Thalidomide treatment of HMECs for 24 h resulted in 60% knockdown of ΔNp63α at the protein level (Fig. S1g, left panel). The cell viability measured by trypan blue staining was more than 90% for both 10 µM and 100 µM thalidomide. In HMECs irradiated at 24 h after thalidomide treatment, CDKN1A mRNA expression increased in a time-dependent manner (P < 0.05) (Fig. S1g, right panel).

ΔNp63α regulates the cell proliferation and apoptosis pathways

We performed nonhierarchical clustering analysis using the k-means method on 500 genes that showed significant variation in RNA-seq data and classified them into two clusters (Fig. 2a). In Cluster A, the expression of each gene was highest in the nonirradiated scr-treated group and lowest in the post-irradiation sip63-treated group, and genes related to cell division were enriched in this cluster (Fig. 2a). This result is consistent with the results described above, where p63 knockdown decreased the expression of genes involved in cell proliferation, and the radiation response further arrested the cell cycle, indicating that ΔNp63α is strongly involved in cell proliferation. On the other hand, Cluster B was enriched in pathways related to cell differentiation, tissue development, cell death, and apoptosis, and the expression of each gene was lowest in the nonirradiated scr-treated group and highest in the post-irradiation sip63-treated group, in contrast to Cluster A (Fig. 2a). These results of enriched pathway analysis are consistent with previous studies reporting that ΔNp63α upregulates genes involved in cell cycle progression, stemness, and stem cell maintenance while downregulating genes involved in apoptosis and cell differentiation [11, 14, 15, 34]. Furthermore, biclustering analysis showed that the expression of genes involved in the cell cycle and stem cell maintenance, such as CDK1/2 and ITGA6/ITGB4, was decreased in the sip63-treated group, while the expression of apoptosis-related genes and tumour suppressor genes, including BAX, TP53, and PTEN, was increased (Fig. 2b and Fig. S3). After irradiation, the expression of CDK inhibitors, such as CDKN1A, and p53-related apoptotic genes, such as BAX and NOXA, was increased (Fig. 2b and Fig. S3). On the other hand, the expression of apoptosis resistance genes, such as Fst and BIRC5, was higher in the scr-treated group than in the sip63-treated group (Fig. 2b and Fig. S1e).

Analysis of the radiation-induced DDR in mammary organoids

In siRNA knockdown experiments, we observed the radiation response of HMECs as described above, but the duration of p63 knockdown was too short, and the experiments were conducted under conditions where the expression of ΔNp63α and the genes that it regulates fluctuated dynamically, so the overall role of ΔNp63α in the radiation response could not be fully dissected. It has been thought that mammary stem cells are present among the basal cells because regenerative mammary glands are produced when mammary basal cells isolated from primary mammary cells by cell sorting are transplanted into rodent mammary fat pads [17, 18]. In addition, it has been recently reported that 3D culture of human and rat mammary basal cells in collagen gels yields mammary organoids that resemble in vivo structures [27, 35]. Therefore, we developed mammary organoids with both ΔNp63α-expressing and non-ΔNp63α-expressing cells by culturing collagen-embedded HMECs and observed the expression of p21 by immunostaining after irradiation. First, single HMECs formed two main types of structures: spherical colonies and mammary organoids (Fig. 3a, b and Fig. S4b). Mammary organoids typically showed structures with a diameter of 1 mm that have branches and acini, similar to the terminal ductal lobular units of mammary epithelium (Fig. 3a). The frequency of mammary organoid formation was approximately 1%. The cells outlining the organoid were positive for ΔNp63α and the basal marker CD49f (Fig. 3b). In general, basal cells of epithelial tissues such as mammary, prostate, and salivary glands strongly express ΔNp63α, and when ΔNp63α is attenuated, they differentiate and show the properties of luminal cells [20]. The organoids produced from a single HMEC in this study consisted of ΔNp63α-positive cells on the outer side and ΔNp63α-negative cells on the inner side, which is consistent with the findings of Centonze et al. [20]. When these organoids were irradiated, the expression of p21 was generally positive in both ΔNp63α-positive and ΔNp63α-negative cells of the acinus region (Fig. 3c and Fig. S4a).

Fig. 3

Radiation-induced DDR of mammary organoids. (a) Whole-mount bright field (BF) and (b) IF imaging of CD49f and p63 (4A4) in the mammary organoids. Scale bars are 50 μm for BF images and 100 μm for IF images. (c) Representative immunohistochemistry images of alveolar tissue in mammary organoids. Red, green, and blue indicate ΔNp63, p21, and DAPI, respectively. Scale bar, 20 μm

Protective role of ΔNp63α against radiation-induced DNA damage

Glutathione peroxidase-2 (GPX2) and cytoglobin (CYGB), which are upregulated by ΔNp63α, have been reported to reduce ROS in cells and thereby protect cells [36,37,38]. In this study, sip63 treatment decreased the expression of GPX2 and CYGB (Figs. 2b and 4a-c), raising a possibility that the sip63 treatment might drastically increase radiation-induced damage itself via decrease of these enzymes with antioxidative functions. The previous experiment showed that ΔNp63α knockdown increased cell responsiveness to radiation, but since the expression of genes with antioxidant effects was also reduced by ΔNp63α knockdown, this effect could also be due to increased DNA oxidative damage, such as DSBs. Therefore, we directly quantified radiation-induced DSBs and intracellular ROS before and after ΔNp63α knockdown. To quantify the radiation-induced DSBs, we performed a neutral comet assay on the siRNA-treated group after irradiation to directly quantify DSBs [39, 40]. The experimental results showed that %tail DNA in both siRNA-treated groups was increased significantly after irradiation (Fig. 4d, P0Gy − 4Gy<0.01). Regardless of irradiation, approximately 5% more %tail DNA was detected in the sip63-treated group than in the scr-treated group, but there was no significant difference between the treatment group. To confirm this result, we further estimated the amount of DSBs from γH2AX foci counts detected by immunostaining. When chromosomal DNA is subjected to radiation to induce DSBs, the H2AX S139 sites around DSBs are phosphorylated by kinases such as ATM; H2AX becomes γH2AX [41, 42]. Therefore, by counting these foci, we can determine the number of DSBs produced by radiation. We counted the γH2AX foci induced in the siRNA-treated groups after radiation, and the number of DSBs generated was compared. After irradiation, γH2AX foci showed a significant increase in both treatment groups (Fig. 4e, P0Gy − 2Gy<0.01). In addition, the sip63-treated group showed a slight increase compared with the scr-treated group (Fig. 4e, P0Gy − 0Gy, 2Gy−2Gy<0.05). To further determine whether ΔNp63α expression affects intracellular ROS, we quantified radiation-induced intracellular ROS using DCFH-DA, which permeates cell membranes and is deacetylated by esterases localized in the cytoplasm, allowing it to react with ROS such as hydrogen peroxide (H2O2) and hydroxyl radicals, resulting in conversion to fluorescent 2’-7’ dichlorofluorescein (DCF) [43]. The experimental results showed that intracellular ROS was increased significantly in both irradiated treatment groups (Fig. 4f, P0Gy − 2 Gy<0.01). Similar to the results described above, intracellular ROS levels in the sip63-treated group were increased by 3% compared to those in the scr-treated group (Fig. 4f, P4Gy − 4 Gy<0.05, P0Gy − 0 Gy=0.07). These experimental results showed that the amounts of DSB and ROS were slightly increased in the sip63-treated group compared to the scr-treated group, indicating that ΔNp63α plays a role in protecting the genomic DNA from oxidative damage by upregulating antioxidant proteins, such as GPX2 and CYGB, and eliminating the intracellular ROS generated by cellular activities. However, it is less potent against radiation that directly generates ROS near DNA in the cell nucleus, indicating that ΔNp63α-mediated antioxidant regulation is not directly involved in controlling the amount of DNA damage caused by radiation.

Fig. 4

Inhibitory effects of GPX2 and CYGB expression on DNA lesions caused by radiation in HMECs. (a) GPX2 and CYGB mRNA expression levels measured by RT–qPCR. Data are the means and SEs of at least three independent assays. (b) Detection of GPX2 and CYGB protein expression in HMECs by Western blotting analysis. (c) The localization of GPX2 and CYGB proteins inside cells. CD49f was used as a HMEC marker. Scale bar, 10 μm. (d) Determination of double-strand breaks (DSBs) using a neutral comet assay. The left panel shows the %tail DNA calculated from the comet tails shown in the right panel (n = 100). (e) Measurement of γH2AX foci observed in the nucleus at 4 h post-irradiation (n = 50). The left panel shows the numbers of γH2AX foci per cell. The right panel shows IF images of γH2AX foci, where green, red, and blue indicate γH2AX, ΔNp63, and DAPI, respectively. In the bottom images, DAPI was used as a counterstaining dye instead of ΔNp63. (f) FCM detection of intercellular reactive oxygen species (ROS) generated in HMECs immediately after X-irradiation. DCFH-DA, one of the major DCF derivatives, and PI were used as probe dyes for detecting ROS and dead cells, respectively. Data are the means and SEs of at least three independent assays. *P < 0.05, **P < 0.01 by Student’s t test

Verification of the DDR inhibitory effect of ΔNp63α by ectopic and entopic expression

ΔNp63α knockdown experiments using HMECs and analysis of organoids revealed that ΔNp63α expression suppresses radiation-induced DDR by inhibiting the transcription of p53-related genes. To verify this, we next observed whether ectopically or entopically expressed ΔNp63α exerted a similar inhibitory effect on the radiation-induced DDR. In this experiment, we used hiPSCs, which are highly susceptible to radiation-induced apoptosis, and generated two types of iPSCs: hiPSCs ectopically expressing ΔNp63α under the Tet-off control (iPS-DN) and hiPSC-derived keratinocytes entopically expressing ΔNp63α (iPS-KC). ΔNp63α expression, which was introduced into hiPSCs by a retroviral vector, was confirmed 3–5 days after Dox removal (Fig. 5a and Fig. S5a). The expression levels of four DDR-related genes, BAX, CDKN1A, NOXA, and GADD45A, in the Dox+ iPS-DNs 24 h post-irradiation were significantly increased compared to those in the nonirradiated cells, while the expression of BAX in the Dox− iPS-DNs was not increased (Fig. 5b). Consistent with this result, protein analysis by Western blotting showed that BAX protein after irradiation was attenuated in Dox− iPS-DNs compared to Dox+ iPS-DNs (Fig. 5e). We also performed ChIP–qPCR using an anti-p53 antibody and examined the change in p53 binding to target sequences before and after ΔNp63α expression. The results showed that p53 in Dox+ iPS-DNs bound directly to both the BAX and CDKN1A promoters post-irradiation, while p53 in Dox− iPS-DNs repressed binding to these promoters (Fig. 5f). FCM analysis showed that the number of apoptotic cells was significantly decreased in Dox− iPS-DNs compared to Dox+ iPS-DNs, which corroborated the results above (Fig. 5d and Fig. S5e). In addition, similar knock-in experiments using human B-cell-derived iPS cells (BiPSC-DN) [44] showed that BAX protein expression after irradiation was attenuated in Dox− BiPSC-DN cells compared to Dox+ BiPSC-DN cells (Fig. S5f-h). On the other hand, cell cycle analysis showed almost the same response in Dox+ iPS-DN and Dox− iPS-DN (Fig. 5c). The measurement of mRNA expression by RT–PCR showed that the increase in CDKN1A expression after irradiation was smaller in Dox− iPS-DN cells than in Dox+ iPS-DN cells, although the difference was not significant (Fig. 5b). The mRNA expression level of GADD45A, which is also transactivated by p53 and involved in G2 phase arrest, was almost the same in Dox+ iPS-DN and Dox− iPS-DN (Fig. 5b). This result was supported by the cell cycle analysis by FCM, which showed that each iPS-DN group underwent cell cycle arrest at G2 or M phase after irradiation (Fig. 5c and Fig. S5d).

Fig. 5

Analyses of iPS-DNs and iPS-KCs expressing ΔNp63α ectopically and entopically, respectively. (a) Immunostaining images of ΔNp63α in iPS-DNs ectopically expressing ΔNp63α with the doxorubicin (Dox) Tet-off system. Scale bar, 20 μm. (b) DDR marker mRNA expression in iPS-DNs post-irradiation. All values were scaled to the expression level of GAPDH as an internal control. Data represent the means and SEs of three independent assays. (c) The frequencies of cell cycle phase in iPS-DN with or without ΔNp63α expression at 24 h after X-irradiation, which were detected by FCM. (d) CC3-positive apoptotic cells detected by FCM in iPS-DNs post-irradiation. (e) Western blotting analysis for iPS-DNs post-irradiation. (f) ChIP analysis of BAX and CDKN1A promoters by anti-p53 antibody in iPS-DNs. Data represent the means of triplicate experiments. Nega: negative control, pro: promoter. (g) Immunostaining images for iPS-KCs entopically expressing ΔNp63α. Scale bar, 20 μm. (h) mRNA expression ratio of Bax and CDKN1A in iPS-KCs post-irradiation (*P < 0.05 vs. 0 Gy by Mann–Whitney U test). (i) Measurement of EdU-positivity frequency in iPS-KCs. (j) Apoptotic cell frequencies in hiPSCs and iPS-KCs at 24 h after irradiation. Data in all figure panels except (h) were analysed with Student’s t test (*P < 0.05, **P < 0.01)

In ectopic expression experiments, ΔNp63α suppressed gene transcription due to p53-activated DDRs, especially for BAX. We further confirmed this result by entopic expression experiments. The differentiation of hiPSCs into human keratinocytes is an established technique used in previous studies [30]. The iPS-KCs differentiated from hiPSCs in this study showed proliferation in a cobblestone-like fashion and expressed hallmarks, including ΔNp63α, CD49f, and CK14, similar to primary human keratinocytes (Fig. 5g and Figs. S5a and S6a, d and e). The CD49f/CD71 ratio, an index of stem cell enrichment in keratinocytes, was comparable between iPS-KCs cultured with collagen I + fibronectin, which has been used as a coating material in previous studies, and those cultured with iMatrix-511, used in this study (Fig. S6a, right panel). After irradiation, BAX and CDKN1A mRNA expression levels were significantly increased in hiPSCs, while in iPS-KCs, CDKN1A showed a significant increase, but BAX showed only a slight increase (Fig. 5h). The frequency of EdU uptake significantly decreased after both 2 and 4 Gy irradiation in hiPSCs but only after 4 Gy irradiation in iPS-KCs (Fig. 5i and Fig. S6b). The number of apoptotic cells detected by FCM showed a significant increase in hiPSCs but not in iPS-KCs (Fig. 5j and Fig. S6c). These FCM results were consistent with those of RNA expression analysis.