When DNA damage responses meet tumor immunity: From mechanism to therapeutic opportunity.

DNA damage is a prevalent phenomenon in the context of cancer progression. Evidence suggests that DNA damage responses (DDR) are pivotal in overcoming tumor immune evasion. Alternatively, traditional radiotherapy and chemotherapy operate by inducing DNA damage, consequently stimulating the immune system to target tumors. The intricate interplay between signaling pathways involved in DDR and immune activation underscores the significance of considering both factors in developing improved immunotherapies. By delving deeper into the mechanisms underlying immune activation brought on by DNA damage, it becomes possible to identify novel treatment approaches that boost the anticancer immune response while minimizing undesirable side effects. This review explores the mechanisms behind DNA damage-induced antitumor immune responses, the importance of DNA damage in antitumor immunity, and potential therapeutic approaches for cancer immunotherapy targeting DDR. Additionally, we discuss the challenges of combination therapy and strategies for integrating DNA damage-targeting therapies with current cancer immunotherapy. In summary, this review highlights the critical role of DNA damage in tumor immunology, underscoring the potential of DDR inhibitors as promising therapeutic modalities for cancer treatment.

Ameliorative effects of zinc and vitamin E against phthalates-induced reproductive toxicity in male rats.

OBJECTIVE: Phthalates (PEs) could cause reproductive harm to males. A mixture of three widely used PEs (MPEs) was used to investigate the ameliorative effects of zinc (Zn) and vitamin E (VE) against male reproductive toxicity. METHODS: Fifty male SD rats were randomly divided into five groups (n = 10). Rats in MPEs group were orally treated with 160 mg/kg/d MPEs, while rats in MPEs combined Zn and/or VE groups were treated with 160 mg/kg/d MPEs plus 25 mg/kg/d Zn and/or 25 mg/kg/d VE. After intervention for 70 days, it's was measured of male reproductive organs' weight, histopathological observation of sperms and testes, serum hormones, PIWI proteins and steroidogenic proteins. RESULTS: Compared with control, anogenital distance, testes weight, epididymides weight, and sex hormones were significantly decreased, while the sperm malformation rate was markedly increased in MPEs group (p < .05); the testicular tissues were injured in MPEs group with disordered and decreased spermatids, and arrested spermatogenesis. PIWIL1, PIWIL2, StAR, CYP11A1 and CYP19A1 were down-regulated in MPEs group (p < .05). However, the alterations of these parameters were restored in MPEs combined Zn and/or VE groups (p < .05). CONCLUSION: Zn and/or VE improved steroid hormone metabolism, and inhibited MPEs' male reproductive toxicity.

Identification of a Novel Gene Signature with DDR and EMT Difunctionalities for Predicting Prognosis, Immune Activity, and Drug Response in Breast Cancer.

Breast cancer, with an overall poor clinical prognosis, is one of the most heterogeneous cancers. DNA damage repair (DDR) and epithelial-mesenchymal transition (EMT) have been identified to be associated with cancer's progression. Our study aimed to explore whether genes with both functions play a more crucial role in the prognosis, immune, and therapy response of breast cancer patients. Based on the Cancer Genome Atlas (TCGA) cancer database, we used LASSO regression analysis to identify the six prognostic-related genes with both DDR and EMT functions, including TP63, YWHAZ, BRCA1, CCND2, YWHAG, and HIPK2. Based on the six genes, we defined the risk scores of the patients and reasonably analyzed the overall survival rate between the patients with the different risk scores. We found that overall survival in higher-risk-score patients was lower than in lower-risk-score patients. Subsequently, further GO and KEGG analyses for patients revealed that the levels of immune infiltration varied for patients with high and low risk scores, and the high-risk-score patients had lower immune infiltration's levels and were insensitive to treatment with chemotherapeutic agents. Furthermore, the Gene Expression Omnibus (GEO) database validated our findings. Our data suggest that TP63, YWHAZ, BRCA1, CCND2, YWHAG, and HIPK2 can be potential genetic markers of prognostic assessment, immune infiltration and chemotherapeutic drug sensitivity in breast cancer patients.

The DDR-related gene signature with cell cycle checkpoint function predicts prognosis, immune activity, and chemoradiotherapy response in lung adenocarcinoma.

BACKGROUND: As a DNA surveillance mechanism, cell cycle checkpoint has recently been discovered to be closely associated with lung adenocarcinoma (LUAD) prognosis. It is also an essential link in the process of DNA damage repair (DDR) that confers resistance to radiotherapy. Whether genes that have both functions play a more crucial role in LUAD prognosis remains unclear. METHODS: In this study, DDR-related genes with cell cycle checkpoint function (DCGs) were selected to investigate their effects on the prognosis of LUAD. The TCGA-LUAD cohort and two GEO external validation cohorts (GSE31210 and GSE42171) were performed to construct a prognosis model based on the least absolute shrinkage and selection operator (LASSO) regression. Patients were divided into high-risk and low-risk groups based on the model. Subsequently, the multivariate COX regression was used to construct a prognostic nomogram. The ssGSEA, CIBERSORT algorithm, TIMER tool, CMap database, and IC50 of chemotherapeutic agents were used to analyze immune activity and responsiveness to chemoradiotherapy. RESULTS: 4 DCGs were selected as prognostic signatures, and patients in the high-risk group had a lower overall survival (OS). The lower infiltration levels of immune cells and the higher expression levels of immune checkpoints appeared in the high-risk group. The damage repair pathways were upregulated, and chemotherapeutic agent sensitivity was poor in the high-risk group. CONCLUSIONS: The 4-DCGs signature prognosis model we constructed could predict the survival rate, immune activity, and chemoradiotherapy responsiveness of LUAD patients.

miR-29b-3p Increases Radiosensitivity in Stemness Cancer Cells via Modulating Oncogenes Axis.

Radioresistance conferred by cancer stem cells (CSCs) is the principal cause of the failure of cancer radiotherapy. Eradication of CSCs is a prime therapeutic target and a requirement for effective radiotherapy. Three dimensional (3D) cell-cultured model could mimic the morphology of cells in vivo and induce CSC properties. Emerging evidence suggests that microRNAs (miRNAs) play crucial roles in the regulation of radiosensitivity in cancers. In this study, we aim to investigate the effects of miRNAs on the radiosensitivity of 3D cultured stem-like cells. Using miRNA microarray analysis in 2D and 3D cell culture models, we found that the expression of miR-29b-3p was downregulated in 3D cultured A549 and MCF7 cells compared with monolayer (2D) cells. Clinic data analysis from The Cancer Genome Atlas database exhibited that miR-29b-3p high expression showed significant advantages in lung adenocarcinoma and breast invasive carcinoma patients' prognosis. The subsequent experiments proved that miR-29b-3p overexpression decreased the radioresistance of cells in 3D culture and tumors in vivo through interfering kinetics process of DNA damage repair and inhibiting oncogenes RBL1, PIK3R1, AKT2, and Bcl-2. In addition, miR-29b-3p knockdown enhanced cancer cells invasion and migration capability. MiR-29b-3p overexpression decreased the stemness of 3D cultured cells. In conclusion, our results demonstrate that miR-29b-3p could be a sensitizer of radiation killing in CSC-like cells via inhibiting oncogenes expression. MiR-29b-3p could be a novel therapeutic candidate target for radiotherapy.

Ilomastat contributes to the survival of mouse after irradiation via promoting the recovery of hematopoietic system.

Ilomastat, a broad-spectrum inhibitor of matrix metalloproteinases (MMPs), has drawn attentions for its function in alleviating radiation damage. However, the detailed mechanisms of Ilomastat's protection from animal model remain not fully clear. In this study, the C57BL/6 mice were pre-administrated with Ilomastat or vihicle for 2 h, and then total body of mice were exposed to 6 Gy of γ-rays. The protective effect of Ilomastat on the hematopoietic system in the irradiated mice were investigated. We found that pretreatment with Ilomastat significantly reduced the level of TGF-ß1 and TNF-α, and elevated the number of bone marrow (BM) mononuclear cells in the irradiated mice. Ilomastat pretreatment also increased the fraction of BM hematopoietic progenitor cells (HPCs) and hematopoietic stem cells (HSCs) at day 30 after irradiation, and protected the spleen of mouse from irradiation. These results suggest that Ilomastat promotes the recovery of hematopoietic injury in the irradiated mice, and thus contributes to the survival of mouse after irradiation.

Suppression of innate immune signaling molecule, MAVS, reduces radiation-induced bystander effect.

PURPOSE: Mitochondrial antiviral signaling (MAVS) protein, located in the mitochondrial out-membrane, is necessary for IFN-beta induction and IFN-stimulated gene expression in response to external stress such as viral invasion and ionizing radiation (IR). Although the involvement of radiation induced bystander effect (RIBE) has been investigated for decades for secondary cancer risk related to radiotherapy, the underlying regulatory mechanisms remain largely unclear, especially the roles played by the immune factors such as MAVS. MATERIAL AND METHODS: MAVS gene knockout cells using CRISPR/Cas9 technology were used as donor cells or recipient cells to assess the role of MAVS in RIBE by means of co-cultured system. The micronucleus and γH2AX foci in the recipient cells were counted to demonstrate the degree of RIBE. The reactive oxygen species (ROS) level in the recipient was measured using the fluorescent dye 2'7'-dichlorofluorescein. RESULTS: Firstly, we found that MAVS expression level was different in A549, BEAS-2B, U937 and HepG2 cells. Cell co-culture experiments showed that MAVS participate in RIBE. Interestingly, the RIBE response was more significant in recipient cells with higher level of MAVS (i.e. A549) than that in recipient cells showing lower level of MAVS (i.e. BEAS-2B). Further, the bystander response was dramatically suppressed in MAVS-silenced A549 and BEAS-2B recipient cells. MAVS-silenced recipient cells exhibited lower level of ROS induced by IR. CONCLUSIONS: Our results indicated that the innate immune signaling molecule MAVS in recipient cells participate in RIBE. ROS is an important factor in RIBE via MAVS pathway and MAVS may be a potential target for the precise radiotherapy and radioprotection.

LET-Dependent Low Dose and Synergistic Inhibition of Human Angiogenesis by Charged Particles: Validation of miRNAs that Drive Inhibition.

Space radiation inhibits angiogenesis by two mechanisms depending on the linear energy transfer (LET). Using human 3D micro-vessel models, blockage of the early motile stage of angiogenesis was determined to occur after exposure to low LET ions (<3 KeV/AMU), whereas inhibition of the later stages occurs after exposure to high LET ions (>8 KeV/AMU). Strikingly, the combined effect is synergistic, detectible as low as 0.06 Gy making mixed ion space radiation more potent. Candidates for bystander transmission are microRNAs (miRNAs), and analysis on miRNA-seq data from irradiated mice shows that angiogenesis would in theory be downregulated. Further analysis of three previously identified miRNAs showed downregulation of their targets associated with angiogenesis and confirmed their involvement in angiogenesis pathways and increased health risks associated with cardiovascular disease. Finally, synthetic molecules (antagomirs) designed to inhibit the predicted miRNAs were successfully used to reverse the inhibition of angiogenesis.

The Reduced Oligomerization of MAVS Mediated by ROS Enhances the Cellular Radioresistance.

Although the mitochondrial antiviral signaling protein (MAVS), located in the mitochondrial outmembrane, is believed to be a signaling adaptor with antiviral feature firstly, it has been shown that suppression of MAVS enhanced radioresistance. The mechanisms underlying this radioresistance remain unclear. Our current study demonstrated that knockdown of MAVS alleviated the radiation-induced mitochondrial dysfunction (mitochondrial membrane potential disruption and ATP production), downregulated the expressions of proapoptotic proteins, and reduced the generation of ROS in cells after irradiation. Furthermore, inhibition of mitochondrial ROS by the mitochondria-targeted antioxidant MitoQ reduced amounts of oligomerized MAVS after irradiation compared with the control group and also prevented the incidence of MN and increased the survival fraction of normal A549 cells after irradiation. To our knowledge, it is the first report to indicate that MAVS, an innate immune signaling molecule, is involved in radiation response via its oligomerization mediated by radiation-induced ROS, which may be a potential target for the precise radiotherapy or radioprotection.

(±)-Alternamgin, a Pair of Enantiomeric Polyketides, from the Endophytic Fungi Alternaria sp. MG1.

A pair of enantiomeric polyketides, (+)- and (-)-alternamgin (1), featuring an unprecedented 6/6/6/6/5/6/6 seven ring backbone, were isolated from the endophytic fungi Alternaria sp. MG1. The relative configuration of 1 was determined using X-ray diffraction, and the absolute configurations of (±)-1 were confirmed by comparing the experimental and calculated ECD data. Plausible biosynthetic pathways for 1 were proposed. Compound (-)-1 exhibited moderate necrosis rates to Hela and HepG2 cells, but (+)-1 only showed similar necrosis rates to HepG2 cells.

miR-222 enhances radiosensitivity of cancer cells by inhibiting the expression of CD47.

Radiotherapy is one of the most common and effective treatments for localized cancer. However, radiotherapy kills tumor cells while causing damage to surrounding normal cells. Enhancing the radiation sensitivity of tumor cells and reducing the radiation damage to normal cells is a difficult problem. Here, we find that the expression of a human microRNA (miRNA), hsa-miR-222, is upregulated in response to ionizing radiation. TargetScan analysis shows that the 3' UTR of CD47 is potentially targeted by miR-222. This prediction was validated by luciferase reporter and mutation assays. It was demonstrated that miR-222 negatively regulates CD47 expression at mRNA and protein levels, and overexpression of the miR-222 enhances cancer cell radiosensitivity by the CD47-pERK pathway in cancer cells. Our findings enrich the complex relationship between miRNA and CD47 in irradiation stress and shed light on the potential of miRNAs both for direct cancer therapeutics and as tools to sensitize tumor cells to radiotherapy.

Toxic effects and foundation of proton radiation on the early-life stage of zebrafish development.

Proton is a major particle of space radiation environment and a prospective radiotherapy beam. However, its risk needs to be fully evaluated for the understanding and to establish the better protective strategy for astronaut and patient. Zebrafish is an ideal model for the toxicity studies on medicines and environmental genetic toxicants. In the current study, embryos of zebrafish at 24 h post-fertilization (hpf) were exposed to proton beam. Some toxic parameters of embryo-larval development were investigated. Microarray combining with qRT-PCR were used to detect the gene expression situation. Generally, fractions of a variety of abnormal phenotypes of embryos and larvae increased in a dose-dependent manner after irradiation. The copy number of mitochondria, the basal respiration rate and the maximum respiration rate of embryos significantly decreased after irradiation. Microarray data demonstrated that MAPK signaling pathway, cell communication, glycolysis and TGF-ß signaling pathway were significantly affected in the irradiated group. The expressions of matrix metallopeptidase 9 (mmp9) and TIMP metallopeptidase inhibitor 2b (timp2b) genes, and enzymatic activity of MMP9 were significantly upregulated in irradiated group. Overall, these results suggest that acute radiation of proton severely affects the development of organism and results in aberration occurrence in the early stage of zebrafish development, which may relates to mitochondrial and glycolytic dysfunction.

Ilomastat, a synthetic inhibitor of MMPs, prevents lung injury induced by γ-ray irradiation in mice.

Lung injury is one of the pathological features in human or animal after radiation and the main side effect for patient after lung cancer radiotherapy. The efficient protective strategy still needs to exploit and the underlying mechanisms remain to be investigated. We found that the expression and activity of matrix metalloproteinases (MMPs) significantly increased at the early stage of radiation-induced lung injury (RILI). Pretreatment with Ilomastat, a synthetic inhibitor of MMPs, decreased the expression and activity of MMPs and significantly alleviated the lung inflammation and fibrosis in the irradiated mice, as well as enhanced the survival of irradiated mice. In addition, the levels of TGF-ß, IL-6, TNF-α and IL-1ß in the tissues dramatically reduced in the irradiated mice pretreated with Ilomastat. Furthermore, our experiments in vitro also showed that radiation significantly increased the MMPs activity, and Ilomastat pretreatment inhibited the activity of MMPs activated by irradiation and increased the cell survival. It is the first report, to our knowledge, to demonstrate that Ilomastat is a potential effective reliever for RILI and MMPs may play important roles in the process of RILI.

Serum microRNAs as Early Indicators for Estimation of Exposure Degree in Response to Ionizing Irradiation.

Exposure to ionizing radiation from nuclear devices, spaceflights or terrorist attacks represents a major threat to human health and public security. After a radiological incident, noninvasive biomarkers that can facilitate rapid assessment of exposure risk in the early stages are urgently needed for optimal medical treatment. Serum microRNAs (miRNAs) are ideal biomarkers because they are stable in response to environmental changes, they are common among different species and are easily collected. Here, we performed miRNA PCR arrays to analyze miRNA expression profiles at 24 h postirradiation. Blood samples were collected from animals that received 0.5-2 Gy total-body carbon-ion irradiation. A specific signature with 12 radiosensitive miRNAs was selected for further validation. After exposure to 0.1-2 Gy of carbon-ion, iron-ion or X-ray radiations, five miRNAs that showed a significant response to these radiation types were selected for further observation of dose- and time-dependent changes: miR-183-5p, miR-9-3p, miR-200b-5p, miR-342-3p and miR-574-5p. We developed a universal model using these five miRNAs to predict the degree of exposure to different radiation types with high sensitivity and specificity. In conclusion, we have identified a set of miRNAs that are quite sensitive to different radiation types in the early stages after exposure, demonstrating their potential use as effective indicators to predict the degree of exposure.

Coroglaucigenin enhances the radiosensitivity of human lung cancer cells through Nrf2/ROS pathway.

Seven cardenolides isolated from the ethanol extract of the stems of Calotropis gigantea were evaluated in vitro against human cancer cells and the structure-activity relationships were discussed. The results demonstrated that a compound, named CGN (coroglaucigenin), had better anti-proliferative activity with the IC50 value less than 6 µM among these compounds. Further, we found that CGN displayed much lower cytotoxicity to normal lung epithelial cells (BEAS-2B) than cancer cells (A549). Especially, our results demonstrated that treatment with CGN (1 µM) combined with X-ray irradiation induced higher radiosensitivity in human lung cancer cells (A549, NCI-H460, NCI-H446) but not in BEAS-2B. The expression levels of nuclear transcription factor Nrf2 and Nrf2-driven antioxidant molecule NQO-1 reduced in A549 cells after combined treatment compared to the radiation only. However, CGN had no toxicity and the levels of antioxidant molecules expression were higher in BEAS-2B cells when given the similar treatment as A549 cells. These results suggest that CGN is a very promising potential sensitizer for cancer radiotherapy, which not only inhibits the proliferation of cancer cells but also enhances the radiosensitivity of cancer cells through suppressing the expression of antioxidant molecules while there is no influence for normal cells.

Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells.

Three dimensional (3D) culture in vitro is a new cell culture model that more closely mimics the physiology features of the in vivo environment and is being used widely in the field of medical and biological research. It has been demonstrated that cancer cells cultured in 3D matrices are more radioresistant compared with cells in monolayer (2D). However, the mechanisms causing this difference remain largely unclear. Here we found that the cell cycle distribution and expression of cell cycle regulation genes in 3D A549 cells are different from the 2D. The higher levels of the promotor methylation of cell cycle regulation genes such as RBL1 were observed in 3D A549 cells compared with cells in 2D. The treatments of irradiation or 5-Aza-CdR activated the demethylation of RBL1 promotor and resulted in the increased expression of RBL1 only in 3D A549 cells. Inhibition of RBL1 enhanced the radioresistance and decreased the G2/M phase arrest induced by irradiation in 2D A549 and MCF7 cells. Overexpression of RBL1 sensitized 3D cultured A549 and MCF7 cells to irradiation. Taken together, to our knowledge, it is the first time to revealthat the low expression of RBL1 due to itself promotor methylation in 3D cells enhances the radioresistance. Our finding sheds a new light on understanding the features of the 3D cultured cell model and its application in basic research into cancer radiotherapy and medcine development.

Radiation induces premature chromatid separation via the miR-142-3p/Bod1 pathway in carcinoma cells.

Radiation-induced genomic instability plays a vital role in carcinogenesis. Bod1 is required for proper chromosome biorientation, and Bod1 depletion increases premature chromatid separation. MiR-142-3p influences cell cycle progression and inhibits proliferation and invasion in cervical carcinoma cells. We found that radiation induced premature chromatid separation and altered miR-142-3p and Bod1 expression in 786-O and A549 cells. Overexpression of miR-142-3p increased premature chromatid separation and G2/M cell cycle arrest in 786-O cells by suppressing Bod1 expression. We also found that either overexpression of miR-142-3p or knockdown of Bod1 sensitized 786-O and A549 cells to X-ray radiation. Overexpression of Bod1 inhibited radiation- and miR-142-3p-induced premature chromatid separation and increased resistance to radiation in 786-O and A549 cells. Taken together, these results suggest that radiation alters miR-142-3p and Bod1 expression in carcinoma cells, and thus contributes to early stages of radiation-induced genomic instability. Combining ionizing radiation with epigenetic regulation may help improve cancer therapies.

Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation.

Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92-1 cells. By measuring senescence associated-ß-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research.

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs.

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of ß-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine.