Published papers

Biliary tract carcinoma (BTC) is a malignancy with poor prognosis. Current molecular targeted therapies benefit only a limited subset of patients, underscoring the need for novel approaches, such as combining DNA-damaging chemoradiotherapy with an immune checkpoint inhibitor. Although cellular responses following chemoradiotherapy-induced DNA damage are essential, they remain poorly understood in the context of three-dimensional tumor structures. This study aimed to investigate the effects of ionizing radiation, a DNA-damaging cancer therapy, on patient-derived BTC organoids. DNA repair and gene expression regulation was integrally examined by immunofluorescence and RNA-Seq analysis following 10 Gy of X-rays. X-ray irradiation caused significant morphological changes. In addition, X-ray irradiation upregulated extracellular matrix-related gene expression as revealed by RNA sequencing. DNA damage response analysis indicated that non-homologous end joining was the primary repair pathway in patient-derived BTC organoids. Moreover, X-ray irradiation activated immune-related pathways, such as cGAS/STING, RIG-I/MDA-5, and JAK/STAT, suggesting potential immune activation following radiotherapy. Our study revealed that DNA damage response altered the tumor structure and modulated the expression of multiple genes, including extracellular matrix- and immune-related genes. Studies using in vitro organoid models can be useful for investigating three-dimensional cellular responses after DNA-damaging cancer therapies, such as chemoradiotherapy.

Malignant solid tumors comprise not only cancer cells but also diverse non-cancerous stromal cells that shape the tumor microenvironment. The tricarboxylic acid (TCA) cycle has an overarching presence in providing substrates needed to drive the electron transport chain and, ultimately, ATP synthesis. However, it remains unclear which stromal cell lineages influence tumor growth through TCA-dependent mitochondrial function, and whether such activities act in a tumor-promoting or tumor-suppressive manner. Isocitrate dehydrogenase 3 (IDH3), a rate-limiting TCA cycle enzyme that generates NADH to support mitochondrial respiration, provides a genetic entry point to interrogate mitochondrial TCA-dependent function in stromal cells. In this study, we established a mouse model in which tamoxifen administration induces CreERT2-dependent knockout of the α subunit of IDH3 (IDH3α) in all somatic cells. Using this model with transplantation of Idh3a-intact murine cancer cells, we found that host Idh3a deficiency accelerated growth of murine MC38 tumors in a cancer cell line-dependent manner. Bone marrow chimera experiments indicated that hematopoietic lineages were not responsible for this phenotype, suggesting a contribution from tissue-resident non-hematopoietic stromal cells that are not replaced by bone marrow transplantation. Single-cell RNA sequencing of human tumor specimens revealed broad IDH3A expression across multiple tumor microenvironment compartments, including fibroblasts. Consistently, in vitro co-culture assays demonstrated that Idh3a-intact, but not Idh3a-KO, fibroblasts suppressed cancer cell proliferation in a contact-dependent manner. Together, these findings identify IDH3α-dependent mitochondrial function in fibroblasts as a critical determinant of tumor progression and suggest that stromal mitochondrial metabolism represents an important axis for modulating tumor behavior.

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.

Purpose: Esophageal cancer lacks characteristic mutations in DNA repair genes; therefore, DNA damage response (DDR) factors have not been widely explored as predictive biomarkers in esophageal cancer. In this study, we explored the potential heterogeneity of DDR capabilities following exposure to ionizing radiation (IR). Methods and Materials: DNA repair protein RAD51 homolog 1/Rad51 recombinase (RAD51), breast cancer susceptibility gene 1, and replication protein A foci formation were analyzed in 15 esophageal cancer cell lines after IR. DNA damage signaling, including phosphorylation of ataxia telangiectasia mutation, Chk2, and Chk1, was examined by immunoblotting. G2/M checkpoint arrest after IR was assessed by scoring mitotic cells. The mode of cell death and cell viability after IR were evaluated using immunofluorescence staining and colony formation assay. Results: Notably, we found significant variations in RAD51 foci formation among 15 esophageal cancer cell lines. Analyzing 2 cell lines, with the highest and lowest RAD51 foci formation each, revealed that cells with low RAD51 foci formation (DDR-defective cell lines) exhibited impaired double-strand break (DSB) end resection, and reduced ataxia telangiectasia mutation-Chk2 and ATR-Chk1 signaling. The DDR-defective cell lines showed increased mitosis with DSBs and enhanced radiosensitivity. Conversely, DDR-proficient cell lines that exhibited intact G2/M checkpoint arrest become significantly more radiosensitive when treated with ATR or WEE1 inhibitors, which abrogate G2/M checkpoint arrest and increase mitosis with DSBs. Conclusions: Esophageal cancer cell lines with lower capability of RAD51 foci formation exhibited defective DDR and G2/M checkpoint arrest associated with higher radiosensitivity. These findings suggest novel possibilities for predicting the efficacy of DNA damage-inducing cancer therapies, such as chemoradiotherapy, based on DDR proficiency, potentially guiding personalized treatment strategies for esophageal cancer.

Purpose: Recent radiation therapy (RT), such as intensity modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT. Methods and Materials: Dermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with 2 normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined. Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR. Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT.