Esophageal Cancer Cells Exhibit Heterogeneity in DNA Double-Strand Break Repair and G2/M Checkpoint Arrest Associated With Cell Viability After Ionizing Radiation.

Introduction
Esophageal cancer is primarily caused by chronic irritation of the esophageal epithelium and is influenced by lifestyle, environmental, and genetic factors.1 Esophageal cancer is recognized as a particularly challenging malignancy owing to its aggressive nature and late-stage diagnosis in many cases.2 Additionally, the anatomic complexity of the esophagus further complicates treatment.3 Moreover, the proximity of cancer to vital structures in the chest and its tendency to metastasize early contribute to poor prognosis.4 Despite advances in treatment, the global 5-year survival rate for esophageal cancer remains low—generally below 20%—which is substantially poorer than that for most other cancers,5 partly due to the lack of effective biomarkers for esophageal cancer treatment.
Radiation therapy and chemotherapy are mainstays of treatment that kill cancer cells by inducing genotoxic stress, particularly DNA double-strand breaks (DSBs), which are recognized as cytotoxic DNA lesions.6 In addition, recent reports have demonstrated better clinical outcomes, in cases of advanced disease when immune checkpoint inhibitors (ICIs), such as nivolumab, pembrolizumab, and ipilimumab, are combined with radiation therapy or chemotherapy, compared with radiation therapy or chemotherapy alone.7,8 However, despite the combination of chemoradiotherapy and ICIs, treatment efficacy in esophageal cancer remains limited. As mentioned above, this is partly because of the lack of effective biomarkers, particularly those linked to DSBs.9 Notably, no clear mutation involving DNA damage response (DDR) has been identified or is available for molecular targeted therapy,10,11 even though DNA damage is typically induced following radiation therapy and chemotherapy.12,13 More than half of the patients with esophageal cancer harbor p53 mutations,14 which lead to a lack of G1/S checkpoint arrest and resistance to canonical apoptotic cell death after chemoradiotherapy.15 Furthermore, the efficacy of combined chemoradiotherapy and ICIs can be influenced by DDR status; specifically, the priming of immune responses can be modulated by the initial DDR response.16,17 Thus, clarifying DDR mechanisms in esophageal cancer cells is critically important.
Radiation therapy directly induces DSBs, whereas genotoxic chemotherapeutic drugs used for the treatment of esophageal cancer indirectly generate DSBs. For example, cisplatin forms both intrastrand and interstrand DNA crosslinks, which can give rise to DSBs when DNA replication encounters these lesions.18
DSBs are repaired by either nonhomologous end joining (NHEJ) or homologous recombination (HR).19,20 The choice of the DSB repair pathway is controlled by cell cycle phase. HR contributes to DSB repair in the S/G2 phase when a DNA template is available following DNA replication. NHEJ functions throughout the cell cycle; that is, even when HR is available, NHEJ also contributes to DSB repair in the S/G2 phase.19,21 DSB end resection, which generates a 3′ single-strand DNA (ssDNA) overhang, is an essential step in HR initiation. MRE11 and CtIP initiate DSB end resection, followed by resection extension using EXO1/DNA2/BLM.22 Additionally, breast cancer susceptibility gene 1 (BRCA1), a breast cancer susceptibility gene, promotes this process via multiple functions.23,24 Subsequently, the generated ssDNA is coated with replication protein A (RPA) and BRCA1 and BRCA2 facilitate the switch from RPA to DNA repair protein RAD51 homolog 1/Rad51 recombinase (RAD51) in ssDNA.25, 26, 27 RAD51-coated ssDNA promotes strand invasion and recombination with sister chromatid templates.28 Thus, HR is categorized as DSB end resection and RAD51-dependent strand invasion/recombination. Alongside DSB repair, DNA damage signaling, for example, cell cycle checkpoint arrest, is activated after DSB induction. As a central kinase in response to DSBs, the ataxia telangiectasia mutation (ATM) triggers signal transduction downstream of DSB repair and cell cycle checkpoint arrest.29 ATM phosphorylates and activates the Chk2 and p53 pathways, resulting in G1/S checkpoint arrest and apoptosis.29 Additionally, in the S/G2 phase, ATM initiates DSB end resection, followed by the activation of ataxia telangiectasia and Rad3-related (ATR) genes during in the HR pathway. Therefore, both ATM and ATR contribute to DSB repair and G2/M checkpoint arrest.30, 31, 32, 33 ATR phosphorylates Chk1, which contributes to the maintenance of G2/M checkpoint arrest and prevents aberrant cell division by blocking mitotic entry.31
In this study, we aimed to address whether esophageal cancer cells show diverse DNA repair capabilities, particularly targeting HR and DNA damage signaling, even in mutation-free tumors. Surprisingly, we found a significant heterogeneity in RAD51 foci formation among 15 esophageal cancer cell lines after ionizing radiation (IR), that is, x-ray irradiation. Additionally, cell lines exhibiting low capability for RAD51 foci formation showed impaired DNA damage signaling leading to a failure of G2/M checkpoint arrest, which is defined as DDR-defective esophageal cancer cell lines. The high frequency of mitotic entry with DSBs in DDR-defective cell lines made them more sensitive to radiation compared to cell lines capable of forming RAD51 foci (DDR-proficient) after x-ray irradiation. Hence, our results provide novel insights into the heterogeneity in DDR among esophageal cancer cell lines which will contribute to the development of strategies to predict prognosis based on DNA repair capability and improve the efficacy of therapies in patients resistant to chemoradiotherapy.

Methods and Materials

Cell culture, irradiation, and treatment
TE1, TE4, TE5, TE9, TE11, TE14, TE15, and ECGI10 cells were obtained from the RIKEN cell bank. KYSE30, KYSE70, KYSE140, KYSE410, KYSE450, and KYSE510 were obtained from Japanese Collection of Research Bioresources Cell Bank.34 The OE21 cells were obtained from KAC Co., Ltd. All cancer cells were cultured in Eagle’s minimum essential medium (#051-07615, FUJIFILM Wako Pure Chemical Corporation) with 10% fetal bovine serum (SERANA) and 1 × Penicillin-Streptomycin-L-Glutamine solution (#161-23201, FUJIFILM Wako Pure Chemical Corporation). The human retinal pigment epithelia-hTERT (Clontech) cell line was cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (SERANA), 1 × Penicillin-Streptomycin-L-Glutamine solution (#161-23201, FUJIFILM Wako Pure Chemical Corporation), and sodium bicarbonate (FUJIFILM Wako Pure Chemical Corporation). All cells were cultured at 37°C with 5% CO2.
X-ray irradiation was performed using MX-160Labo (160 kVp, 1.07 Gy/min, 3.00 mA, MediXtec). The ATR inhibitor (ceralasertib) (#S7693, Selleck Chemicals), WEE1 inhibitor (adavosertib) (#S1525, Selleck Chemicals) and the RAD51 inhibitor (B02) (#553525, Calbiochem) were dissolved in dimethyl sulfoxide. All inhibitors were adjusted at a concentration of 10 mM and stored at -20 °C. Thirty minutes before x-ray irradiation, the inhibitors were added to the culture medium. The medium was unchanged throughout the experiments.

Immunofluorescence staining
Cells were seeded onto cover glasses (Matsunami). Cells in G2 or S phase were identified using CENPF staining, while cells in S phase were identified using 5-ethynil-2′-deoxyuridine (EdU) staining. EdU (10 μM) was added 30 minutes before x-ray irradiation. At indicated time points, cells were fixed for 10 minutes in 3% paraformaldehyde–2% sucrose and permeabilized for 3 minutes with 0.2% TritonX-100 in phosphate-buffered saline (PBS). Cells were washed twice with PBS and incubated at 37°C for 30 minutes with the primary antibody in 2% bovine serum albumin in PBS. Cells were then washed twice with PBS and incubated at 37°C for 30 minutes with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 in 2% bovine serum albumin in PBS. Subsequently, cells were stained with Click-iT EdU. After an additional wash with PBS, the coverslips were mounted on glass slides using ProLong Gold Antifade Reagent with 4,6-diamidino-2-phenylindole (DAPI) (#8961; Cell Signaling Technology).

Antibodies
The primary antibodies used in this study are listed in the Table E1. The secondary antibodies anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (1:500, #4408, Cell Signaling Technology), anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (1:500, #8890, Cell Signaling Technology), anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (1:500, #8889, Cell Signaling Technology) and anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (1:500, #4412, Cell Signaling Technology), antirat IgG (H + L), (Alexa Fluor 488 Conjugate) (1:500, #4416, Cell Signal Technology) were used in immunofluorescence staining and anti-rabbit IgG, horseradish peroxidase-linked antibody (1:4000, #7074; Cell Signaling Technology), anti-mouse IgG, horseradish peroxidase-linked antibody (1:4000, #7076; Cell Signal Technology) were used in immunoblotting. EdU staining was performed using the Click-iT EdU Cell Proliferation Kit for Imaging and Alexa Fluor 647 dye (Thermo Fisher Scientific).

Foci counting
X-ray-induced foci were observed using a Nikon ECLIPSE Ni microscope equipped with a 100 × /1.45 numerical aperture oil immersion objective lens, a DS-Qi2 camera, and NIS-Elements D imaging software (Nikon). Cells were co-stained with CENPF and EdU and CENPF-positive-EdU-negative cells were categorized as G2 phase cells. Only G2 phase cells were included in the counting. Foci were blindly scored as described previously.21,35 γH2AX foci were quantified 30 minutes after 0.5 Gy to capture the peak number of DNA DSBs before significant repair occurred, as reported in multiple previous studies.21,36 Higher doses yielded too many γH2AX foci for reliable quantification at this time point; thus, 0.5 Gy was selected. RAD51 foci were quantified 2 hours after 2 Gy to evaluate HR activity, because lower doses produced too few RAD51 foci to allow reliable quantification at this time point.

Immunoblotting
Cells were harvested using 1 × sample buffer (250 mM Tris pH6.8, 10% sodium dodecyl sulfate, 30% glycerol, 5% 3-mercapto-1,2-propanediol, 0.02% bromophenol blue) following a wash with PBS. The cell lysate was sonicated, followed by heat treatment at 95°C for 5 min. The cell lysates were immediately subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories). Subsequently, the proteins were transferred onto a nitrocellulose membrane, and the transfer was confirmed by Ponceau-S staining. The membrane was then washed with 1 × Tris buffered saline containing 0.05% Tween 20 (TBST). Thereafter, the membrane was blocked for 1 hour followed by incubation with the primary antibody for >16 hours at room temperature. After washing with 1 × TBST, the membrane was incubated with the secondary antibody reaction for 1 hour at room temperature. Thereafter, the membranes were incubated with enhanced chemiluminescence reagents after washing with 1 × TBST. Chemiluminescence was detected using an Amersham Imager 600. The intensity of each band was measured using ImageJ software (version 1.54 g). The ratio of the phosphorylated protein to total protein was calculated by normalizing the intensities of the phosphorylated protein bands to the intensity of total protein. These values ​​were further normalized to those obtained under nonirradiated conditions.

G2/M checkpoint analysis
Exponentially growing cells were irradiated on glass coverslips. Cells were stained with histone H3 (pS10) and DAPI, and histone H3 (pS10)-positive cells with condensed chromatin were counted as mitotic cells. More than 400 cells were counted in randomly selected fields of view. The percentage of mitotic cells was calculated, and the mitotic index was calculated as the value normalized to the percentage of mitotic cells under nonirradiated conditions.31

Classification of cell death mode
Cells were irradiated and fixed with paraformaldehyde at the indicated time points after x-ray irradiation, and immunofluorescence staining was performed. Cleaved caspase-3 staining positive cells were classified as apoptotic.37 Cells harboring micronuclei or multinucleated cells were counted. A total of 200 cells were counted for each condition.

Single sample gene set enrichment analysis
Single sample gene set enrichment analysis (ssGSEA),38 an extension of the GSEA method was performed using the R package “GSVA” to analyze the mRNA expression of DNA repair-related gene sets. The gene expression profile was obtained from the Cancer Cell Line Encyclopedia (CCLE) RNA-seq data set (Expression_Public_23Q2, Broad Institute; https://depmap.org/portal/).39 For each of the 15 esophageal cancer cell lines analyzed, ssGSEA enrichment scores were calculated and subsequently normalized to z-scores within each gene set to facilitate comparisons among cell lines.

Correlation analysis between radiosensitivity and RAD51 ratio
Radiosensitivity data using ATP bioluminescence assay were obtained from a previously published study.40 Correlation analysis between radiosensitivity and the RAD51 ratio in 14 esophageal cancer cell lines was performed.

Colony formation assay
Cells were seeded in 35 mm dishes. After overnight incubation, inhibitors at the indicated concentrations were added 30 minutes before radiation irradiation. The cells were then irradiated with 0, 2, 4, or 6 Gy x-rays. The media was changed 48 hours after inhibitor addition. After 10 days of incubation, cells were washed in PBS and treated with crystal violet solution consisting of 0.5% crystal violet (#038-04862, FUJIFILM Wako Pure Chemical Corporation), 20% methanol, and H2O for 20 min. After washing the dishes to remove the crystal violet solution, the colonies were observed using a Kenis LZ-LED-B stereomicroscope (Kenis). Colonies containing more than 50 cells were counted. The plating efficiency (PE) was calculated as the number of colonies formed divided by the number of seeded cells. The surviving fraction was calculated as the PE for each condition divided by the PE of the control (nonirradiated).41

Statistical analysis
Bar, violin, and dot plots were created using the R package ggplot2 (version 3.5.1). Pearson’s correlation coefficient was calculated using the R package ggpubr (version 0.6.0). The numbers of independent experiments are shown in the figure legends. Statistical significance was determined using the 2-tailed Student’s t-test or Welch’s t-test. If the Shapiro–Wilk test failed, the Mann-Whitney U test was used. The Bonferroni correction was applied to avoid a high rate of family-wise errors for data sets in which multiple comparisons were performed using a single group. The significance levels are shown in each panel as follows: *P < .05, **P < .01, ***P < .001; n.s., not significant.

Results

Substantial heterogeneity in DNA DSB repair capability in esophageal cancer cell lines
RAD51 foci formation, a marker of HR repair, was examined after x-ray irradiation to investigate the DNA repair capability in esophageal cancer (N.B., x-ray was used because the timing of DSB induction is readily controlled by x-rays as opposed to that with chemotherapeutic drugs). In this study, we selected 15 esophageal cancer cell lines whose gene expression and mutation status are available in the CCLE database. RAD51 foci formation was examined 2 hours after x-ray irradiation, a time point shown to represent the peak of RAD51 foci formation in nonmalignant cells of previous studies (Fig. 1A, B and Fig. E1A).21,42 Notably, we found substantial heterogeneity in x-ray irradiation-induced RAD51 foci formation among esophageal cancer cell lines (Fig. 1B). The number of γH2AX foci 30 minutes after x-ray irradiation also exhibited diversity (Fig. 1C, D and Fig. E1B). In this study, RAD51 and γH2AX foci in the G2 phase were examined using the cell cycle markers CENPF and EdU (Fig. S2A). Subsequently, the number of RAD51 foci at 2 hours after x-ray irradiation (indicating HR activity) was normalized to the number of γH2AX foci at 30 minutes after x-ray irradiation (indicating DSB induction by x-ray) (Fig. 1E). Analysis of normalized values confirmed substantial heterogeneity in the RAD51 ratio (RAD51/γH2AX), suggesting that DSB repair capability differs between esophageal cancer cell lines.
As we used esophageal cancer cell lines whose RNA-seq information is available in the CCLE database, the levels of mRNA expression and mutation status of DSB repair genes were investigated (Fig. E2B). Notably, no apparent correlation was observed between HR gene expression and the x-ray irradiation-induced RAD51 foci ratio. In addition, the expression of NHEJ genes was examined; however, no significant correlation was observed. Furthermore, no correlation was observed between DNA damage signal transduction genes and RAD51 ratio. Similar to the mRNA expression results, no representative mutations in common genes that correlated with the RAD51 ratio were identified (Fig. E2B). We further analyzed DSB repair gene expression profile using the ssGSEA method and compared normalized enrichment scores among 15 cell lines (Fig. E2C). However, there was no apparent correlation between the enrichment scores of HR-, NHEJ-, or DSB signal transduction-related gene sets and the RAD51 ratio. Thus, our DDR foci analysis revealed substantial heterogeneity in HR capability, that is, DSB repair heterogeneity, in esophageal cancer cells. In contrast, it was difficult to predict x-ray irradiation-induced DSB repair using the mRNA expression of repair genes.

Impaired DSB end resection and DNA damage signaling are associated with the low RAD51 ratio phenotype in esophageal cancer cells
As demonstrated in Fig. 1, we found significant heterogeneity in RAD51 foci formation between esophageal cancer cell lines. Therefore, we selected 2 cell lines from the top and bottom of the RAD51 ratio: TE1 and ECGI10 (low RAD51 ratio), and KYSE450 and KYSE510 (high RAD51 ratio) and examined DSB end resection, a central step in HR, and the activation of signal transduction following DSB induction.20,43
As a first step in HR repair, DSB ends are resected using DNA nucleases.19 BRCA1 is a critical factor that promotes DSB end resection in combination with several other resection factors such as MRE11, CtIP, and EXO1.19 To investigate the ability of BRCA1 recruitment at DSB sites, BRCA1 foci were examined after x-ray irradiation. Notably, we found that esophageal cancer cell lines exhibiting a low RAD51 ratio showed impaired BRCA1 foci formation after x-ray irradiation (Fig. 2A, B, Fig. E3A), suggesting low resection activity at DSB sites. Consistent with the results for BRCA1 foci, RPA foci, a marker of DSB end resection, were also impaired in esophageal cancer cell lines that exhibited a low RAD51 ratio (Fig. 2C, D, Fig. E3B). These data suggest that low RAD51 foci formation is a consequence of poor DSB end resection activity in these cell lines.
Thereafter, the phosphorylation of ATM, Chk2, and Chk1 was examined in TE1 (low RAD51 ratio) and KYSE510 (high RAD51 ratio) cells to determine whether the level of RAD51 ratio is associated with DNA damage-induced signal transduction. We observed a minimal increase in the phosphorylation of ATM and Chk2 in TE1 cells (low RAD51 ratio) (Fig. 2E). In addition, the phosphorylation of Chk1 was minimal after x-ray irradiation (Fig. 2F; a comparison between 2 Gy and 6 Gy in each cell line is shown in Fig. E3C, D). In contrast, a clear increase in ATM, Chk2, and Chk1 phosphorylation was observed in KYSE510 cells (high RAD51 ratio) after x-ray irradiation (Fig. 2E, F; the quantification of the signal intensity is shown in Fig. 2G).
ATM is required for DSB end resection,21 which promotes RPA and BRCA1 foci formation, resulting in activation of ATR/Chk1 signaling and RAD51 loading onto ssDNA. Therefore, the observed phenotypes, namely, decreased DDR, impaired DSB end resection, and reduced RAD51 loading in TE1 cells after x-ray irradiation, appeared consistent. Accordingly, cells exhibiting low RAD51 ratios (TE1 and ECGI10) and high RAD51 ratios (KYSE450 and KYSE510) were defined as DDR-defective and DDR-proficient cell lines, respectively.

DDR-defective esophageal cancer cell lines fail G2/M checkpoint arrest, causing mitotic entry with excessive DSBs and abnormal nuclear morphology
ATM-Chk2 and ATR-Chk1 signal transduction contributes to cell cycle checkpoint arrest, such as G2/M and G1/S checkpoint arrest.20,43 G1/S checkpoint arrest was likely impaired in the esophageal cancer cells used in this study because they harbored p53 mutations (Fig. E2B). In contrast, because G2/M checkpoint arrest is highly dependent on the ATM and ATR pathway, rather than p53, cells are generally arrested in G2 phase after x-rays.31 As shown in Fig. 2, esophageal cancer cell lines exhibiting low RAD51 ratios (TE1 and ECGI10) showed poor ATM-Chk2 and ATR-Chk1 signaling after x-ray irradiation; therefore, we examined the activity of G2/M checkpoint arrest by monitoring the progression of mitotic entry after x-ray irradiation. Notably, we found mitotic cells in DDR-defective cell lines (TE1 and ECGI10) at 2 to 4 hours after x-ray irradiation, suggesting an incomplete G2/M checkpoint arrest, although partial G2/M arrest was observed. In contrast, DDR-proficient cell lines (KYSE450 and KYSE510) exhibited nearly complete arrest at 2 to 4 hours after x-ray irradiation (Fig. 3A; statistical analysis is shown in E2). To confirm whether mitotic cells escaped from the initiation of G2/M checkpoint arrest harbor DSBs or not, cells were stained with histone H3 pSer10 and γH2AX, which are mitosis and DSB markers, respectively. Notably, we found a substantial number of DSBs in the mitotic cells of DDR-defective cell lines (TE1 and ECGI10) (Fig. 3B, C). At 8 hours after irradiation, when DDR-proficient cell lines restart mitosis, the number of γH2AX foci in mitotic cells was examined. As predicted, the number of γH2AX foci in DDR-proficient cell lines was lower than that in DDR-defective cell lines (Fig. 3B, C, and Fig. E4A-C).
Mitosis harboring a substantial number of DSBs is likely to cause mitotic catastrophe after x-rays. Therefore, we quantified the number of cells displaying abnormal nuclear morphology (eg, multinucleation or micronucleation), which may eventually undergo mitotic catastrophe,44 for up to 96 hours after x-ray irradiation. Strikingly, we observed a significant increase in such cells in DDR-defective cell lines (TE1 and ECGI10) compared with DDR-proficient cell lines (KYSE450 and KYSE510) (Fig. 3D, E, statistical analysis is shown in Table E3). Consistent with previous reports demonstrating poor apoptosis induction in a p53-deficient background after x-ray irradiation, only a few apoptotic cells were detected in both cell lines. In addition, we examined the number of micronuclei and found that TE1 cells exhibiting a high number of micronuclei, particularly those with more than 10 micronuclei, displayed abnormal nuclear morphology resembling mitotic catastrophe (Fig. 3F).
Taken together, these data suggest that the failure of G2/M checkpoint arrest in DDR-defective cell lines causes mitotic entry with excessive DSBs, leading to mitotic catastrophe, which is a representative mode of cell death in p53-negative cancer cells.

DDR-defective esophageal cancer cell lines exhibit greater radiosensitivity
HR repair is one of the major DSB repair pathways in human cells following x-rays.19 Because HR is used in the S/G2 phase of cycling cells, its dependency increases with higher proliferation rates, that is, HR deficiency causes greater radiosensitivity in actively proliferating cancer cells. Therefore, to investigate the relationship between DDR activity and survival rate after IR, a correlation analysis was conducted using a data set regarding radiosensitivity in cancer cell lines using an ATP bioluminescence assay.40 The x-ray irradiation-induced ratio of RAD51 foci obtained in this study was positively correlated with radiosensitivity, although this was not statistically significant. Notably, DDR-defective cell lines (TE1 and ECGI10) exhibited greater radiosensitivity, whereas the top RAD51 ratio and DDR-proficient cell lines (KYSE450 and KYSE510) showed radioresistance (Fig. 4A). In contrast, the RAD51 mRNA expression obtained using CCLE did not correlate with radiosensitivity (Fig. E5A), supporting the notion that it is difficult to predict DSB repair capability using the mRNA expression of repair genes. Subsequently, a colony formation assay was performed after x-ray irradiation of DDR-defective cell lines (TE1 and ECGI10) and DDR-proficient cell lines (KYSE450 and KYSE510) to further confirm the correlation between the RAD51 ratio and radiosensitivity. The DDR-defective cell lines (TE1 and ECGI10) showed greater radiosensitivity than the DDR-proficient cell lines (KYSE450 and KYSE510) (Fig. 4B, Fig. E5B, statistical analysis is shown in Table E4). These data confirm that radiosensitivity is enhanced under DDR deficiency. Thus, our analysis demonstrated heterogeneity in DDR activity among esophageal cancer cell lines, although representative pathogenic mutations in the DDR genes were not observed in esophageal cancer. This heterogeneity is associated with radiosensitivity and may affect the efficacy of radiation therapy and DNA damage-based chemotherapy.

Radiosensitivity in DDR-proficient esophageal cancer cell line is augmented by the inhibition of G2/M checkpoint arrest
Next, to determine which process in DDR (DNA damage signaling and repair) affect the enhancement of radiosensitivity, that is, whether G2/M checkpoint arrest, or RAD51 foci formation, radiosensitivity was examined with or without ATR, WEE1, or RAD51 inhibitor (N.B., WEE1 is also involved in G2/M checkpoint arrest, by phosphorylating and inactivating cyclin-dependent kinase 1 following DNA damage).33 To investigate whether ATR, WEE1, or RAD51 influences G2/M checkpoint arrest, mitotic index after x-rays was examined in the presence of either ATR, WEE1, or RAD51 inhibitor. As expected, both ATR and WEE1 inhibition impaired G2/M checkpoint arrest (Fig. 5A), causing severe DSB formation in mitotic cells (Fig. 5B, C). In contrast, RAD51 inhibition did not affect G2/M checkpoint arrest, which is consistent with the notion that RAD51 is recruited following the activation of ATR/Chk1 following resection; that is, the lack of RAD51 activity does not significantly affect the upstream events of ATR/Chk1 signal transduction and G2/M checkpoint arrest. Finally, radiosensitivity was examined using a colony formation assay in the presence of ATR, WEE1, or RAD51 inhibitors. Notably, ATR or WEE1 inhibition increased radiosensitivity, whereas RAD51 inhibition had less of an impact (Fig. 5D-F).
Taken together, these results suggest that the combination of irradiation and inhibition of the G2/M checkpoint is a promising strategy, especially for radioresistant esophageal cancer.

Discussion
In this study, we demonstrated substantial heterogeneity in the DNA repair capabilities among esophageal cancer cell lines. Unlike breast and ovarian cancers, characteristic mutations in DNA repair genes have not been reported in esophageal cancer; therefore, DNA repair capacity or DNA damage signaling after chemoradiotherapy is presumed not to significantly differ among esophageal cancers. In addition, owing to the absence of mutations in DNA repair genes, significant differences in DNA damage signaling following chemoradiotherapy are not expected in esophageal cancer. However, in the present study, we found substantial heterogeneity in DNA repair capacity and signaling pathways in esophageal cancer cells, which was notably correlated with differences in the efficacy of G2/M checkpoint arrest following IR, that is, x-ray irradiation. Specifically, DDR-defective esophageal cancer cells exhibited impaired DSB end resection and G2/M checkpoint signaling, which are associated with abnormal nuclear morphology and radiosensitivity. These findings suggest novel possibilities for predicting the efficacy of DNA damage-inducing cancer therapies, such as chemoradiotherapy, based on DDR activity, and potentially guiding personalized treatment strategies for esophageal cancer.
Previous studies have demonstrated that esophageal cancer lacks characteristic mutations in key DNA repair genes; therefore, it has been challenging to establish effective strategies for targeted therapies.45 Contrary to this understanding, our analysis revealed substantial heterogeneity in both DNA repair and signaling, that is, the existence of DDR-defective esophageal cancer, independent of identifiable DDR gene mutations. This novel observation contrasts sharply with previous reports, suggesting uniformity in DNA repair capabilities owing to the absence of identifiable mutations in DDR-related genes. Moreover, these findings propose a possibility that similar hidden heterogeneity may also exist in other cancers, which are not currently applicable for DDR targeting cancer therapy.46,47 Furthermore, our selection of the RAD51 foci assay as a primary marker for DDR analysis was experimentally advantageous because RAD51 antibodies reliably produced clear, distinct foci compared to other DDR markers, such as BRCA1 or RPA, which are not commonly used in clinical samples. Therefore, RAD51 foci assays could represent a robust tool not only for esophageal cancer, but also for the first screening of DDR heterogeneity across multiple cancer types and for further translational validation in clinical samples.
Our study further highlights that DDR impairment in esophageal cancer cells is strongly associated with defective G2/M checkpoint arrest. Particularly, we observed that cells defective in RAD51 foci formation (DDR-defective TE1 cells) failed to properly activate the ATM-Chk2 and ATR-Chk1 signaling pathways, resulting in unsuccessful G2/M checkpoint arrest. Consequently, these cells progressed to mitosis while harboring unrepaired DSBs, ultimately resulting in an increased number of cells exhibiting abnormal nuclear morphology. This observation underscores the fact that radiosensitivity in DDR-defective cells is driven by compromised G2/M checkpoint arrest. Supporting this mechanism, we confirmed that pharmacological inhibition of ATR or WEE1, key mediators of G2/M arrest, dramatically sensitized DDR-proficient esophageal cancer cells to x-ray irradiation. Conversely, the inhibition of RAD51 alone had a limited impact on radiosensitivity, suggesting that the effective induction of cell death post radiation relies predominantly on disrupting the G2/M checkpoint arrest. Therefore, although RAD51 foci assay is suitable for DDR analysis as mentioned above, we stress that impaired RAD51 foci formation does not necessarily lead to a failure of the G2/M checkpoint arrest, because RAD51 functions downstream of DSB end resection and ATR/Chk1 activation. Thus, a suitable marker should be selected in each purpose.
Taken together, our findings demonstrate previously unrecognized heterogeneity in DDR among esophageal cancer cell lines, challenging the notion that DDR status can be inferred solely from genetic analysis, that is, genomic and transcriptome approaches. However, although RAD51 foci formation serves as a robust experimental marker for identifying DDR-defective cells, as demonstrated in this study, further investigation of G2/M checkpoint proteins or combined DDR markers is required to accurately predict therapeutic responses. Future studies should validate these findings using clinical samples and relevant animal models such as patient-derived xenografts.
This study has several limitations. Both γH2AX and RAD51 foci were examined at a single radiation dose and time point (30 minutes after 0.5 Gy and 2 hours after 2 Gy, respectively). These time points were selected because they correspond to the peaks of foci formation in nonmalignant cells, as shown in previous studies.21,36,42 However, we cannot exclude the possibility of cell line- or dose-dependent differences in kinetics. Another limitation is the absence of direct analyses of DSB repair, such as reporter assays or comet assays. Consequently, our conclusions regarding heterogeneity in DDR signaling are based on correlative evidence. Future studies incorporating multiple doses, time points, and direct repair assays will be necessary to fully characterize this heterogeneity.
Notably, prior research has already suggested RAD51 and γH2AX as biomarkers for chemoradiotherapy responsiveness.48, 49, 50 These reports indicate potential clinical applicability of DDR factors, particularly when DNA damage inducing cancer therapy, that is, chemoradiotherapy, is applied. Thus, our findings provide important insights into the previously unrecognized heterogeneity of DNA repair capabilities and signaling pathways highlighting their significance as predictive biomarkers potentially targeting ATM, ATR, Chk1, and RAD51 of therapeutic response to chemoradiotherapy.