Details of a Researcher - Shibata, Atsushi

Papers - Shibata, Atsushi

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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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Carbon ion radiation and clustered DNA double-strand breaks

Atsushi Shibata

DNA Damage and Double Strand Breaks - Part A (Elsevier) 117 - 130 2022

ISSN 1874-6047

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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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Genome Maintenance Mechanisms at the Chromatin Level

Hirotomo Takatsuka, Atsushi Shibata, Masaaki Umeda

International Journal of Molecular Sciences (MDPI AG) 22 ( 19 ) 10384 - 10384 2021.09

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.

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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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ATM’s Role in the Repair of DNA Double-Strand Breaks

Atsushi Shibata, Penny A. Jeggo

Genes (MDPI AG) 12 ( 9 ) 1370 - 1370 2021.08

Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage—e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair.

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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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高LET線ならびに放射線類似薬により誘発されるDNA二本鎖切断の修復における修復経路の重要度の解析(Requirement of DNA double-strand break repair pathways for repairing high LET radiation- and radiomimetic agents-induced DNA damage)

黒沢綾, 高橋昭久, 武田茂樹, 柴田淳史, 足立典隆

日本放射線影響学会大会講演要旨集 ((一社)日本放射線影響学会) 63回 75 - 75 2020.10

ISSN 1347-8680

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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Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair

Atsushi Shibata, Penny A Jeggo

Journal of Radiation Research (Oxford University Press (OUP)) 61 ( 5 ) 718 - 726 2020.09

ISSN 0449-3060

<title>Abstract</title>
p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

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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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Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks

Atsushi Shibata, Penny A. Jeggo

DNA Repair (Elsevier BV) 93 102915 - 102915 2020.09

ISSN 1568-7864

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Reporting of methodologies used for clonogenic assays to determine radiosensitivity

Takahiro Oike, Shuichiro Komatsu, Yuka Komatsu, Ankita Nachankar, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno

Journal of Radiation Research (Oxford University Press (OUP)) 61 ( 6 ) 828 - 831 2020.08

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

<title>Abstract</title>
Radiotherapy treatment strategies should be personalized based on the radiosensitivity of individual tumors. Clonogenic assays are the gold standard method for in vitro assessment of radiosensitivity. Reproducibility is the critical factor for scientific rigor; however, this is reduced by insufficient reporting of methodologies. In reality, the reporting standards of methodologies pertaining to clonogenic assays remain unclear. To address this, we performed a literature search and qualitative analysis of the reporting of methodologies pertaining to clonogenic assays. A comprehensive literature review identified 1672 papers that report the radiosensitivity of human cancer cells based on clonogenic assays. From the identified papers, important experimental parameters (i.e. number of biological replicates, technical replicates, radiation source and dose rate) were recorded and analyzed. We found that, among the studies, (i) 30.5% did not report biological or technical replicates; (ii) 47.0% did not use biological or technical replicates; (iii) 3.8% did not report the radiation source; and (iv) 32.3% did not report the dose rate. These data suggest that reporting of methodologies pertaining to clonogenic assays in a considerable number of previously published studies is insufficient, thereby threatening reproducibility. This highlights the need to raise awareness of standardization of the methodologies used to conduct clonogenic assays.

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