Details of a Researcher - Shibata, Atsushi

Papers - Shibata, Atsushi

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ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair

Nho Cong Luong, Hidemasa Kawamura, Hiroko Ikeda, Reiko T. Roppongi, Atsushi Shibata, Jiaxuan Hu, Jinmeng G. Jiang, David S. Yu, Kathryn D. Held

International Journal of Radiation Biology (Taylor & Francis group) 1 - 12 2024

Research paper (scientific journal), Joint Work, Accepted

Purpose
Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines.

Methods
Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage.

Results
Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells.

Conclusion
Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

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Characterization of the signal transduction cascade for inflammatory gene expression in fibroblasts with ATM-ATR deficiencies after Ionizing radiation

Shunji Haruna, Ken Okuda, Akiko Shibata, Mayu Isono, Kohei Tateno, Hiro Sato, Takahiro Oike, Yuki Uchihara, Yu Kato, Atsushi Shibata

Radiotherapy and Oncology (Elsevier) 194 110198 2024

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Background and purpose
Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR.
Materials and methods
Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor–based downstream gene expression mapping (TDEM) method developed in this study.
Results
Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3–pathway between ATM and ATR deficiency.
Conclusion
Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.

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Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation

Ryota Hayashi, Hikaru Okumura, Mayu Isono, Motohiro Yamauchi, Daiki Unami, Rahmartani Tania Lusi, Masamichi Yamamoto, Yu Kato, Yuki Uchihara, Atsushi Shibata

Journal of Radiation Research (Oxford University Press) 2024

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5′-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

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Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks

Kie Ozaki, Reona Kato, Takaaki Yasuhara, Yuki Uchihara, Miyako Hirakawa, Yu Abe, Hiroki Shibata, Reika Kawabata-Iwakawa, Aizhan Shakayeva, Palina Kot, Kiyoshi Miyagawa, Keiji Suzuki, Naoki Matsuda, Atsushi Shibata & Motohiro Yamauchi

Scientific Reports 14(1):18455 2024

Accepted

Comprehensive single cell analysis demonstrates radiotherapy-induced infiltration of macrophages expressing immunosuppressive genes into tumour in oesophageal squamous cell carcinoma

Hidekazu Oyoshi, Junyan Du, Shunsuke A Sakai, Riu Yamashita, Masayuki Okumura, Atsushi Motegi, Hidehiro Hojo, Masaki Nakamura, Hidenari Hirata, Hironori Sunakawa, Daisuke Kotani, Tomonori Yano, Takashi Kojima, Yuka Nakamura, Motohiro Kojima, Ayako Suzuki, Junko Zenkoh, Katsuya Tsuchihara, Tetsuo Akimoto, Atsushi Shibata, Yutaka Suzuki, Shun-Ichiro Kageyama

Science Advances (American Association for the Advancement of Science) 9 ( 50 ) 2023.12

Research paper (scientific journal), Joint Work, Corresponding author, Accepted

Radiotherapy (RT) combined with immunotherapy is promising; however, the immune response signature in the clinical setting after RT remains unclear. Here, by integrative spatial and single-cell analyses using multiplex immunostaining (CODEX), spatial transcriptome (VISIUM), and single-cell RNA sequencing, we substantiated the infiltration of immune cells into tumors with dynamic changes in immunostimulatory and immunosuppressive gene expression after RT. In addition, our comprehensive analysis uncovered time- and cell type–dependent alterations in the gene expression profile after RT. Furthermore, myeloid cells showed prominent up-regulation of immune response–associated genes after RT. Notably, a subset of infiltrating tumor-associated myeloid cells showing PD-L1 positivity exhibited significant up-regulation of immunostimulatory (HMGB1 and ISG15), immunosuppressive (SIRPA and IDO1), and protumor genes (CXCL8, CCL3, IL-6, and IL-1AB), which can be targets of immunotherapy in combination with PD-L1. These datasets will provide information on the RT-induced gene signature to seek an appropriate target for personalized immunotherapy combined with RT and guide the timing of combination therapy.

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HLA Class I Expression Is Associated with DNA Damage and Immune Cell Infiltration into Dysplastic and Neoplastic Lesions in Ulcerative Colitis.

Okami H, Ozawa N, Sohda M, Yokobori T, Osone K, Erkhem-Ochir B, Dorjkhorloo G, Shiraishi T, Okada T, Sano A, Sakai M, Miyazaki T, Ogawa H, Yao T, Oike T, Sato H, Shirabe K, Shibata A, Saeki H

International journal of molecular sciences (MDPI) 24 ( 17 ) 13648 2023.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 1661-6596

Human leukocyte antigen class I (HLA-I) is considered a genetic pathogen for ulcerative colitis (UC). This study aimed to investigate the significance of DNA damage and HLA-I expression in infiltrating immune cells and immune checkpoint protein PD-L1 expression in dysplasia/colitic cancer (CC) and sporadic colorectal cancer (SCRC). We performed immunohistochemical staining for HLA-I, PD-L1, γH2AX (DNA damage marker), and immune cell markers such as CD8, FOXP3, CD68, and CD163 (in surgically resected specimens from 17 SCRC patients with 12 adjacent normal mucosa (NM) and 9 UC patients with 18 dysplasia/CC tumors. The ratio of membrane HLA-I-positive epithelial cells in UC and dysplasia/CC tissues was significantly higher than that in NM and SCRC. High HLA-I expression in dysplasia/CC was associated with high positivity of γH2AX and PD-L1 expression compared to SCRC. The infiltration of CD8-positive T cells and CD68-positive macrophages in HLA-I-high dysplasia/CC was significantly higher than in UC and SCRC. Dysplasia/CC specimens with DNA damage exhibited high levels of HLA-I-positive epithelial cells with high CD8- and CD68-positive immune cell infiltration compared to UC and SCRC specimens. Targeting DNA damage in UC may regulate immune cell infiltration, immune checkpoint proteins, and carcinogenesis by modulating DNA damage-induced HLA-I antigen presentation.

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Analysis of the relationship between LET, γH2AX foci volume and cell killing effect of carbon ions using high-resolution imaging technology

Takahiro Oike, Sangeeta Kakoti, Makoto Sakai, Akihiko Matsumura, Tatsuya Ohno, Atsushi Shibata

Journal of Radiation Research (Oxford University Press (OUP)) 64 ( 2 ) 335 - 344 2023.03

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 0449-3060

Abstract

The strong cell killing effect of high linear energy transfer (LET) carbon ions is dependent on lethal DNA damage. Our recent studies suggest that induction of clusters of double-strand breaks (DSBs) in close proximity is one of the potential mechanisms. However, the relationship between LET, the degree of DSB clustering and the cell killing effect of carbon ions remains unclear. Here, we used high-resolution imaging technology to analyze the volume of γH2AX foci induced by monoenergetic carbon ions with a clinically-relevant range of LET (13–100 keV/μm). We obtained data from 3317 γH2AX foci and used a gaussian function to approximate the probability (p) that 1 Gy-carbon ions induce γH2AX foci of a given volume (vth) or greater per nucleus. Cell killing effects were assessed in clonogenic assays. The cell killing effect showed high concordance with p at vth = 0.7 μm3 across various LET values; the difference between the two was 4.7% ± 2.2%. This relationship was also true for clinical carbon ion beams harboring a mixed LET profile throughout a spread-out Bragg peak width (30–120 mm), with the difference at vth = 0.7 μm3 being 1.6% ± 1.2% when a Monte Carlo simulation-derived dose-averaged LET was used to calculate p. These data indicate that the cell killing effect of carbon ions is predictable by the ability of carbon ions to induce γH2AX foci containing clustered DSBs, which is linked to LET, providing the biological basis for LET modulation in the planning of carbon ion radiotherapy.

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Calreticulin Upregulation in Cervical Cancer Tissues From Patients After 10 Gy Radiation Therapy

Kohei Okada, Hiro Sato, Takuya Kumazawa, Yasumasa Mori, Tiara Bunga Mayang Permata, Yuki Uchihara, Shin-ei Noda, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Soehartati Gondhowiardjo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Advances in Radiation Oncology (Elsevier BV) 8 ( 3 ) 101159 2022.12

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 2452-1094

Purpose: Understanding the immune response during radiation therapy (RT) in a clinical setting is imperative for maximizing the efficacy of combined RT and immunotherapy. Calreticulin, a major damage-associated molecular pattern that is exposed on the cell surface after RT, is presumed to be associated with the tumor-specific immune response. Here, we examined changes in calreticulin expression in clinical specimens obtained before and during RT and analyzed its relationship with the density of CD8+ T cells in the same patient set. Methods and Materials: This retrospective analysis evaluated 67 patients with cervical squamous cell carcinoma who were treated with definitive RT. Tumor biopsy specimens were collected before RT and after 10 Gy irradiation. Calreticulin expression in tumor cells was evaluated via immunohistochemical staining. Subsequently, the patients were divided into 2 groups according to the level of calreticulin expression, and the clinical outcomes were compared. Finally, the correlation between calreticulin levels and density of stromal CD8+ T cells was evaluated. Results: The calreticulin expression significantly increased after 10 Gy (82% of patients showed an increase; P <.01). Patients with increased calreticulin levels tended to show better progression-free survival, but this was not statistically significant (P =.09). In patients with high expression of calreticulin, a positive trend was observed between calreticulin and CD8+ T cell density, but the association was not statistically significant (P =.06). Conclusions: Calreticulin expression increased after 10 Gy irradiation in tissue biopsies of patients with cervical cancer. Higher calreticulin expression levels are potentially associated with better progression-free survival and greater T cell positivity, but there was no statistically significant relationship between calreticulin upregulation and clinical outcomes or CD8+ T cell density. Further analysis will be required to clarify mechanisms underlying the immune response to RT and to optimize the RT and immunotherapy combination approach.

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DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production.

Yuki Uchihara, Tiara Bunga Mayang Permata, Hiro Sato, Reika Kawabata-Iwakawa, Sayako Katada, Wenchao Gu, Sangeeta Kakoti, Motohiro Yamauchi, Reona Kato, Soehartati Gondhowiardjo, Naoki Hosen, Takaaki Yasuhara, Atsushi Shibata

Molecular cell (Cell Press) 82 ( 14 ) 2557 - 2570 2022.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 10972765

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

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Radiosensitization by the Selective Pan-FGFR Inhibitor LY2874455.

Narisa Dewi Maulany Darwis, Eisuke Horigome, Shan Li, Akiko Adachi, Takahiro Oike, Atsushi Shibata, Yuka Hirota, Tatsuya Ohno

Cells (MDPI) 11 ( 11 ) 2022.06

Research paper (scientific journal), Joint Work, Accepted

Ionizing radiation activates cytoprotective pathways in cancer cells. Fibroblast growth factor receptor (FGFR) is a key player in these pathways. Thus, FGFR signaling is a potential target to induce radiosensitization. LY2874455 is an orally administrable selective pan-FGFR inhibitor. However, the radiosensitizing effects of LY2874455 remain unclear. In this study, we addressed this issue by using radioresistant human cancer cell lines H1703 (FGFR1 mutant), A549 (FGFR1-4 wild-type), and H1299 (FGFR1-4 wild-type). At an X-ray dose corresponding to 50%-clonogenic survival as the endpoint, 100 nM LY2874455 increased the sensitivity of H1703, A549, and H1299 cells by 31%, 62%, and 53%, respectively. The combination of X-rays and LY2874455 led to a marked induction of mitotic catastrophe, a hallmark of radiation-induced cell death. Furthermore, combination treatment suppressed the growth of A549 xenografts to a significantly greater extent than either X-rays or the drug alone without noticeable toxicity. This is the first report to show the radiosensitizing effect of a selective pan-FGFR inhibitor. These data suggest the potential efficacy of LY2874455 as a radiosensitizer, warranting clinical validation.

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RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

Takaaki Yasuhara, Reona Kato, Motohiro Yamauchi, Yuki Uchihara, Lee Zou, Kiyoshi Miyagawa, Atsushi Shibata

Cell Reports (Cell Press) 38 ( 5 ) 110335 - 110335 2022.02

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 2211-1247

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

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Expression of non‑homologous end joining factor, Ku80, is negatively correlated with PD‑L1 expression in cancer cells after X‑ray irradiation

Takuya Kumazawa, Yasumasa Mori, Hiro Sato, Tiara Permata, Yuki Uchihara, Shin-Ei Noda, Kohei Okada, Sangeeta Kakoti, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Soehartati Gondhowiardjo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Oncology Letters (Spandidos Publications) 23 ( 1 ) 29 - 29 2022.01

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 17921074

The growing importance of antitumour immunity by cancer immunotherapy has prompted studies on radiotherapy-induced immune response. Previous studies have indicated that programmed cell death-1 ligand (PD-L1) expression is regulated by DNA damage signalling. However, PD-L1 up-regulation after radiotherapy has not been fully investigated at the clinical level, particularly in the context of expression of DNA repair factors. The present study examined the correlation of mRNA expression between PD-L1 and non-homologous end joining (NHEJ) factors using The Cancer Genome Atlas database analysis. Among NHEJ factors, Ku80 mRNA expression was negatively correlated with PD-L1 mRNA expression levels in several types of cancer (colon adenocarcinoma, breast invasive carcinoma, skin cutaneous melanoma, lung adenocarcinoma, head and neck squamous cell carcinoma, uterine corpus endometrial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma). To verify the negative correlation in clinical samples, the present study analysed whether Ku80 expression levels affected PD-L1 up-regulation after radiotherapy using cervical squamous cell carcinoma samples. Quantitative evaluation using software analysis of immunohistochemically stained slides revealed that patients with low Ku80 positivity in biopsy specimens demonstrated increased PD-L1 expression levels after 10 Gy irradiation (Spearman's rank correlation coefficient=-0.274; P=0.017). Furthermore, PD-L1 induction levels in tumour cells after 10 Gy of irradiation were significantly inversely correlated with Ku80 expression levels (Spearman's rank correlation coefficient=-0.379; P<0.001). The present study also confirmed that short interfering RNA-mediated Ku80 depletion was associated with greater X-ray-induced PD-L1 up-regulation in HeLa cells. These results indicated that radiotherapy could enhance PD-L1 induction in tumour cells with low Ku80 expression in a clinical setting. Furthermore, these data highlighted Ku80 as a potential predictive biomarker for immune checkpoint therapy combined with radiotherapy.

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64Cu-ATSM Predicts Efficacy of Carbon Ion Radiotherapy Associated with Cellular Antioxidant Capacity

Ankita Nachankar, Takahiro Oike, Hirofumi Hanaoka, Ayaka Kanai, Hiro Sato, Yukari Yoshida, Hideru Obinata, Makoto Sakai, Naoto Osu, Yuka Hirota, Akihisa Takahashi, Atsushi Shibata, Tatsuya Ohno

Cancers (MDPI AG) 13 ( 24 ) 6159 - 6159 2021.12

Research paper (scientific journal), Joint Work, Accepted

Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy.

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Mechanism of chromosome rearrangement arising from single-strand breaks

Palina Kot, Takaaki Yasuhara, Atsushi Shibata, Miyako Hirakawa, Yu Abe, Motohiro Yamauchi, Naoki Matsuda

Biochemical and Biophysical Research Communications (Elsevier BV) 572 191 - 196 2021.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.

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High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells

Tiara Bunga Mayang Permata, Hiro Sato, Wenchao Gu, Sangeeta Kakoti, Yuki Uchihara, Yukihiko Yoshimatsu, Itaru Sato, Reona Kato, Motohiro Yamauchi, Keiji Suzuki, Takahiro Oike, Yoshito Tsushima, Soehartati Gondhowiardjo, Tatsuya Ohno, Takaaki Yasuhara, Atsushi Shibata

Journal of Radiation Research (Oxford University Press (OUP)) 62 ( 5 ) 773 - 781 2021.09

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 04493060

<title>Abstract</title>
Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

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Quantitative volumetric analysis of the Golgi apparatus following X-ray irradiation by super-resolution 3D-SIM microscopy

Takahiro Oike, Yuki Uchihara, Tiara Bunga Mayang Permata, Soehartati Gondhowiardjo, Tatsuya Ohno, Atsushi Shibata

Medical Molecular Morphology (Springer Science and Business Media LLC) 54 ( 2 ) 166 - 172 2021.06

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 18601480

<title>Abstract</title>To obtain quantitative volumetric data for the Golgi apparatus after ionizing radiation (IR) using super-resolution three-dimensional structured illumination (3D-SIM) microscopy. Normal human retinal pigment epithelial (RPE) cells were irradiated with X-rays (10 Gy), followed by immunofluorescence staining of the Golgi marker RCAS1. 3D-SIM imaging was performed using DeltaVision OMX version 4 and SoftWoRx 6.1. Polygon rendering and spot signal identification were performed using Imaris 8.1.2. Differences between groups were assessed by Welch’s <italic>t</italic> test. RCAS1 signals in untreated cells were located adjacent to nuclei and showed a reticular morphology. Upon IR, the area of RCAS1 signals expanded while retaining the reticular morphology. Polygon rendering imaging revealed that the volume of RCAS1 at 48 h post-IR was greater than that for unirradiated cells (93.7 ± 19.0 μm3 vs. 33.0 ± 4.2 μm3, respectively; <italic>P</italic> < 0.001): a 2.8-fold increase. Spot signal imaging showed that the number of RCAS1 spot signals post-IR was greater than that for unirradiated cells [3.4 ± 0.8 (× 103) versus 1.3 ± 0.2 (× 103), respectively; <italic>P</italic> < 0.001]: a 2.7-fold increase. This is the first study to report quantitative volumetric data of the Golgi apparatus in response to IR using super-resolution 3D-SIM microscopy.

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Analysis of radiotherapy‑induced alteration of CD8+ T cells and PD‑L1 expression in patients with uterine cervical squamous cell carcinoma

Yasumasa Mori, Hiro Sato, Takuya Kumazawa, Tiara Permata, Yuya Yoshimoto, Kazutoshi Murata, Shin-Ei Noda, Takuya Kaminuma, Ken Ando, Takahiro Oike, Noriyuki Okonogi, Kohei Okada, Sangeeta Kakoti, Keiji Suzuki, Hayato Ikota, Hideaki Yokoo, Takashi Nakano, Tatsuya Ohno, Atsushi Shibata

Oncology Letters (Spandidos Publications) 21 ( 6 ) 446 - 446 2021.06

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 17921074

Radiotherapy induces an immune response in the cancer microenvironment that may influence clinical outcome. The present study aimed to analyse the alteration of CD8+ T-cell infiltration and programmed death-ligand 1 (PD-L1) expression following radiotherapy in clinical samples from patients with uterine cervical squamous cell carcinoma. Additionally, the current study sought to analyse the association between these immune responses and clinical outcomes. A total of 75 patients who received either definitive chemoradiotherapy or radiotherapy were retrospectively analyzed. CD8+ T-cell infiltration and PD-L1 expression were determined by immunohistochemistry using biopsy specimens before radiotherapy (pre-RT) and after 10 Gy radiotherapy (post-10 Gy). The PD-L1+ rate was significantly increased from 5% (4/75) pre-RT to 52% (39/75) post-10 Gy (P<0.01). Despite this increase in the PD-L1+ rate post-10 Gy, there was no significant association between both pre-RT and post-10 Gy and overall survival (OS), locoregional control (LC) and progression-free survival (PFS). On the other hand, the CD8+ T-cell infiltration density was significantly decreased for all patients (median, 23.1% pre-RT vs. 16.9% post-10 Gy; P=0.038); however, this tended to increase in patients treated with radiotherapy alone (median, 17.7% pre-RT vs. 24.0% post-10 Gy; P=0.400). Notably, patients with high CD8+ T-cell infiltration either pre-RT or post-10 Gy exhibited positive associations with OS, LC and PFS. Thus, the present analysis suggested that CD8+ T-cell infiltration may be a prognostic biomarker for patients with cervical cancer receiving radiotherapy. Furthermore, immune checkpoint inhibitors may be effective in patients who have received radiotherapy, since radiotherapy upregulated PD-L1 expression in cervical cancer specimens.

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Comparison of Clonogenic Survival Data Obtained by Pre- and Post-Irradiation Methods

Takahiro Oike, Yuka Hirota, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno

Journal of Personalized Medicine (MDPI AG) 10 ( 4 ) 171 - 171 2020.10

Research paper (scientific journal), Joint Work, Accepted

Clonogenic assays are the gold standard to measure in vitro radiosensitivity, which use two cell plating methods, before or after irradiation (IR). However, the effect of the plating method on the experimental outcome remains unelucidated. By using common cancer cell lines, here we demonstrate that pre-IR and post-IR plating methods have a negligible effect on the clonogenic assay-derived photon sensitivity as assessed by SF2, SF4, SF6, SF8, D10, or D50 (N.B. SFx indicates the survival at X Gy; Dx indicates the dose providing X% survival). These data provide important biological insight that supports inter-study comparison and integrated analysis of published clonogenic assay data regardless of the plating method used.

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DNA Repair and Signaling in Immune-Related Cancer Therapy

Sangeeta Kakoti, Hiro Sato, Siddhartha Laskar, Takaaki Yasuhara, Atsushi Shibata

Frontiers in Molecular Biosciences (Frontiers Media SA) 7 205 - 205 2020.09

Research paper (scientific journal), Last author, Corresponding author, Accepted

Cancer therapy using immune checkpoint inhibitors (ICIs) is a promising clinical strategy for patients with multiple types of cancer. The expression of programmed cell death ligand-1 (PD-L1), an immune-suppressor ligand, in cancer cells is a factor that influences the efficacy of ICI therapy, particularly in the anti-programmed cell death protein-1 (PD-1)/PD-L1 antibody therapy. PD-L1 expression in cancer cells are associated with tumor mutation burden including microsatellite instability because the accumulation of mutations in the cancer genome can produce abnormal proteins via mutant mRNAs, resulting in neoantigen production and HLA-neoantigen complex presentation in cancer cells. HLA-neoantigen presentation promotes immune activity within tumor environment; therefore, known as hot tumor. Thus, as the fidelity of DNA repair affects the generation of genomic mutations, the status of DNA repair and signaling in cancer cells can be considered prior to ICI therapy. The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA) database analysis showed that tumor samples harboring mutations in any non-homologous end joining, homologous recombination, or DNA damage signaling genes exhibit high neoantigen levels. Alternatively, an urgent task is to understand how the DNA damage-associated cancer treatments change the status of immune activity in patients because multiple clinical trials on combination therapy are ongoing. Recent studies demonstrated that multiple pathways regulate PD-L1 expression in cancer cells. Here, we summarize the regulation of the immune response to ICI therapy, including PD-L1 expression, and also discuss the potential strategies to improve the efficacy of ICI therapy for poor responders from the viewpoint of DNA damage response before or after DNA damage-associated cancer treatment.

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Induction of Micronuclei in Cervical Cancer Treated with Radiotherapy

Daijiro Kobayashi, Takahiro Oike, Kazutoshi Murata, Daisuke Irie, Yuka Hirota, Hiro Sato, Atsushi Shibata, Tatsuya Ohno

Journal of Personalized Medicine (MDPI AG) 10 ( 3 ) 110 - 110 2020.09

Research paper (scientific journal), Joint Work, Accepted

Micronuclei (MN) trigger antitumor immune responses via the cyclic GMP-AMP synthase-signaling effector stimulator of interferon genes (cGAS-STING) pathway. Radiotherapy induces MN in peripheral blood lymphocytes. However, data for solid tumors are lacking. Here, we analyzed MN post-radiotherapy in solid tumor samples. Tumor biopsy specimens were obtained from seven prospectively recruited patients with cervical cancer, before treatment and after receiving radiotherapy at a dose of 10 Gy (in five fractions). The samples were stained with 4′,6-diamidino-2-phenylindole dihydrochloride, and 200 nuclei per sample were randomly identified and assessed for the presence of MN or apoptosis, based on nuclear morphology. The median number of MN-harboring nuclei was significantly greater in samples from patients treated with radiotherapy than in pre-treatment samples (151 (range, 16–327) versus 28 (range, 0–61); p = 0.015). No significant differences in the number of apoptotic nuclei were observed between pre-treatment and 10 Gy samples (5 (range, 0–30) versus 12 (range, 2–30); p = 0.30). This is the first report to demonstrate MN induction by radiotherapy in solid tumors. The results provide clinical evidence of the activation of antitumor immune responses by radiotherapy.

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