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Nature Communications Jun 2024Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR),...
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RAS oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RAS. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RAS induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
Topics: Humans; Exodeoxyribonucleases; Phosphoproteins; MRE11 Homologue Protein; Signal Transduction; Cellular Senescence; DNA Replication; DNA Damage; Fibroblasts; Interferon-beta
PubMed: 38926338
DOI: 10.1038/s41467-024-49740-w -
Frontiers of Medicine Jun 2024Synthetic lethality is a novel model for cancer therapy. To understand the function and mechanism of BEN domain-containing protein 4 (BEND4) in pancreatic cancer, eight...
Synthetic lethality is a novel model for cancer therapy. To understand the function and mechanism of BEN domain-containing protein 4 (BEND4) in pancreatic cancer, eight cell lines and a total of 492 cases of pancreatic neoplasia samples were included in this study. Methylation-specific polymerase chain reaction, CRISPR/Cas9, immunoprecipitation assay, comet assay, and xenograft mouse model were used. BEND4 is a new member of the BEN domain family. The expression of BEND4 is regulated by promoter region methylation. It is methylated in 58.1% (176/303) of pancreatic ductal adenocarcinoma (PDAC), 33.3% (14/42) of intraductal papillary mucinous neoplasm, 31.0% (13/42) of pancreatic neuroendocrine tumor, 14.3% (3/21) of mucinous cystic neoplasm, 4.3% (2/47) of solid pseudopapillary neoplasm, and 2.7% (1/37) of serous cystic neoplasm. BEND4 methylation is significantly associated with late-onset PDAC (> 50 years, P < 0.01) and tumor differentiation (P < 0.0001), and methylation of BEND4 is an independent poor prognostic marker (P < 0.01) in PDAC. Furthermore, BEND4 plays tumor-suppressive roles in vitro and in vivo. Mechanistically, BEND4 involves non-homologous end joining signaling by interacting with Ku80 and promotes DNA damage repair. Loss of BEND4 increased the sensitivity of PDAC cells to ATM inhibitor. Collectively, the present study revealed an uncharacterized tumor suppressor BEND4 and indicated that methylation of BEND4 may serve as a potential synthetic lethal marker for ATM inhibitor in PDAC treatment.
PubMed: 38926248
DOI: 10.1007/s11684-023-1053-3 -
Anticancer Research Jul 2024The aim of this study was to develop an enhanced intestinal toxicity assay with three outputs assessing proliferation, villi morphology and DNA damage after irradiation.
BACKGROUND/AIM
The aim of this study was to develop an enhanced intestinal toxicity assay with three outputs assessing proliferation, villi morphology and DNA damage after irradiation.
MATERIALS AND METHODS
Whole 5 cm jejunal lengths were collected from mice following total body x-ray irradiation (0-15 Gy) at 0-84 h. Tissues were wrapped into swirls for cryopreservation and immunohistochemically stained for EdU, CD31, and γH2AX. A semi-automated image analysis was developed for the proliferation, villi morphology, and DNA damage models.
RESULTS
Proliferation assessed via EdU staining varied with cycles of damage repair, hyperproliferation, and homeostasis after radiation, with the time to onset of each cycle variable based on radiation dose. An analysis model evaluating the amount of proliferation per unit length of jejunum analyzed was developed, with a dose-response curve identified at 48 h post treatment. The villi length model measured the length of intact and healthy CD31-stained capillary beds between the crypts and villi tips at 3.5 days post treatment within a 0-10 Gy dose range. The DNA damage model evaluated the intensity of γH2AX staining within cellular nuclei, with a useful dose-response identified at 1 h post-radiation treatment.
CONCLUSION
This assay demonstrates flexibility for assessing radiation-induced damage, with analysis of proliferation, villi length, or direct DNA damage achievable at defined time points and within useful radiation dose curves. The software-assisted image analysis allows for rapid, comprehensive, and objective data generation with an assay turnover time of days instead of weeks on samples that are representative of most of the treated jejunum.
Topics: Animals; Mice; Cell Proliferation; DNA Damage; Jejunum; Radiation Tolerance; Intestinal Mucosa; Intestines; Whole-Body Irradiation; Dose-Response Relationship, Radiation; Histones; Male; Mice, Inbred C57BL
PubMed: 38925821
DOI: 10.21873/anticanres.17091 -
The Journal of Molecular Diagnostics :... Jun 2024Replication-coupled gene editing using locked-nucleic-acid-modified single-stranded oligodeoxyribonucleotides (LMOs) can genetically engineer mammalian cells with high...
Replication-coupled gene editing using locked-nucleic-acid-modified single-stranded oligodeoxyribonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, we developed oligonucleotide-directed mutation screening (ODMS) to determine whether variants of uncertain clinical significance of DNA mismatch-repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine (6TG)-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas previously mouse embryonic stem cells (mESCs) hemizygous for DNA mismatch-repair (MMR) genes were used, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6TG resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggested that the efficiency of LMO-mediated gene targeting at a non-selectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.
PubMed: 38925454
DOI: 10.1016/j.jmoldx.2024.05.011 -
Cancer Letters Jun 2024In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery,...
In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.
PubMed: 38925361
DOI: 10.1016/j.canlet.2024.217063 -
Biomedicine & Pharmacotherapy =... Jun 2024Chemotherapy resistance typically leads to tumour recurrence and is a major obstacle to cancer treatment. Increasing numbers of circular RNAs (circRNAs) have been... (Review)
Review
Chemotherapy resistance typically leads to tumour recurrence and is a major obstacle to cancer treatment. Increasing numbers of circular RNAs (circRNAs) have been confirmed to be abnormally expressed in various tumours, where they participate in the malignant progression of tumours, and play important roles in regulating the sensitivity of tumours to chemotherapy drugs. As exosomes mediate intercellular communication, they are rich in circRNAs and exhibit a specific RNA cargo sorting mechanism. By carrying and delivering circRNAs, exosomes can promote the efflux of chemotherapeutic drugs and reduce intracellular drug concentrations in recipient cells, thus affecting the cell cycle, apoptosis, autophagy, angiogenesis, invasion and migration. The mechanisms that affect the phenotype of tumour stem cells, epithelial-mesenchymal transformation and DNA damage repair also mediate chemotherapy resistance in many tumours. Exosomal circRNAs are diagnostic biomarkers and potential therapeutic targets for reversing chemotherapy resistance in tumours. Currently, the rise of new fields, such as machine learning and artificial intelligence, and new technologies such as biosensors, multimolecular diagnostic systems and platforms based on circRNAs, as well as the application of exosome-based vaccines, has provided novel ideas for precision cancer treatment. In this review, the recent progress in understanding how exosomal circRNAs mediate tumour chemotherapy resistance is reviewed, and the potential of exosomal circRNAs in tumour diagnosis, treatment and immune regulation is discussed, providing new ideas for inhibiting tumour chemotherapy resistance.
PubMed: 38925018
DOI: 10.1016/j.biopha.2024.117027 -
DNA Repair Jun 2024Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind...
Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind DNA and organize the genome into chromatin. Post-translational modifications of histones have been shown to play an essential role in orchestrating the chromatin response to DNA damage by regulating the DNA damage response pathway. Among the histone modifications that contribute to this intricate network, histone ADP-ribosylation (ADPr) is emerging as a pivotal component of chromatin-based DNA damage response (DDR) pathways. In this review, we survey how histone ADPr is regulated to promote the DDR and how it impacts chromatin and other histone marks. Recent advancements have revealed histone ADPr effects on chromatin structure and the regulation of DNA repair factor recruitment to DNA lesions. Additionally, we highlight advancements in technology that have enabled the identification and functional validation of histone ADPr in cells and in response to DNA damage. Given the involvement of DNA damage and epigenetic regulation in human diseases including cancer, these findings have clinical implications for histone ADPr, which are also discussed. Overall, this review covers the involvement of histone ADPr in the DDR and highlights potential future investigations aimed at identifying mechanisms governed by histone ADPr that participate in the DDR, human diseases, and their treatments.
PubMed: 38924925
DOI: 10.1016/j.dnarep.2024.103711 -
International Immunopharmacology Jun 2024Harmine has many pharmacological activities and has been found to significantly inhibit the fibrosis of keloid fibroblasts. DNA damage repair (DDR) is essential to...
BACKGROUND
Harmine has many pharmacological activities and has been found to significantly inhibit the fibrosis of keloid fibroblasts. DNA damage repair (DDR) is essential to prevent fibrosis. This study aimed to investigate the effects of harmine on pulmonary fibrosis and its underlying mechanisms.
METHODS
Bleomycin and TGF-β1 were used to construct pulmonary fibrosis models in vivo and in vitro, then treated with harmine to explore harmine's effects in treating experimental pulmonary fibrosis and its related mechanisms. Then, RNA sequencing was applied to investigate further the crucial DDR-related genes and drug targets of harmine against pulmonary fibrosis. Finally, the expression levels of DDR-related genes were verified by real-time quantitative PCR (RT-qPCR) and western blot.
RESULTS
Our in vivo experiments showed that harmine treatment could improve weight loss and lung function and reduce tissue fibrosis in mice with pulmonary fibrosis. The results confirmed that harmine could inhibit the viability and migration of TGF-β1-induced MRC-5 cells, induce their apoptosis, and suppress the F-actin expression, suggesting that harmine could suppress the phenotypic transition from lung fibroblasts to lung myoblasts. In addition, RNA sequencing identified 1692 differential expressed genes (DEGs), and 10 DDR-related genes were screened as critical DDR-related genes. RT-qPCR and western blotting showed that harmine could down-regulate the expression of CHEK1, ERCC1, ERCC4, POLD1, RAD51, RPA1, TOP1, and TP53, while up-regulate FEN1, H2AX and GADD45α expression.
CONCLUSIONS
Harmine may inhibit pulmonary fibrosis by regulating DDR-related genes and activating the TP53-Gadd45α pathway.
PubMed: 38924867
DOI: 10.1016/j.intimp.2024.112542 -
Advanced Science (Weinheim,... Jun 2024Obstructive sleep apnea syndrome (OSAS), characterized by chronic intermittent hypoxia (CIH), is an independent risk factor for aggravating non-alcoholic steatohepatitis...
Obstructive sleep apnea syndrome (OSAS), characterized by chronic intermittent hypoxia (CIH), is an independent risk factor for aggravating non-alcoholic steatohepatitis (NASH). The prevailing mouse model employed in CIH research is inadequate for the comprehensive exploration of the impact of CIH on NASH development due to reduced food intake observed in CIH-exposed mice, which deviates from human responses. To address this issue, a pair-feeding investigation with CIH-exposed and normoxia-exposed mice is conducted. It is revealed that CIH exposure aggravates DNA damage, leading to hepatic fibrosis and inflammation. The analysis of genome-wide association study (GWAS) data also discloses the association between Eepd1, a DNA repair enzyme, and OSAS. Furthermore, it is revealed that CIH triggered selective autophagy, leading to the autophagic degradation of Eepd1, thereby exacerbating DNA damage in hepatocytes. Notably, Eepd1 liver-specific knockout mice exhibit aggravated hepatic DNA damage and further progression of NASH. To identify a therapeutic approach for CIH-induced NASH, a drug screening is conducted and it is found that Retigabine dihydrochloride suppresses CIH-mediated Eepd1 degradation, leading to alleviated DNA damage in hepatocytes. These findings imply that targeting CIH-mediated Eepd1 degradation can be an adjunctive approach in the treatment of NASH exacerbated by OSAS.
PubMed: 38924647
DOI: 10.1002/advs.202405955 -
Advanced Science (Weinheim,... Jun 2024Ferroptosis is a form of regulated cell death that can be modulated by small molecules and has the potential for the development of therapeutics for oncology. Although...
Ferroptosis is a form of regulated cell death that can be modulated by small molecules and has the potential for the development of therapeutics for oncology. Although excessive lipid peroxidation is the defining hallmark of ferroptosis, DNA damage may also play a significant role. In this study, a potential mechanistic role for MIF in homologous recombination (HR) DNA repair is identified. The inhibition or genetic depletion of MIF or other HR proteins, such as breast cancer type 1 susceptibility protein (BRCA1), is demonstrated to significantly enhance the sensitivity of cells to ferroptosis. The interference with HR results in the translocation of the tumor suppressor protein p53 to the mitochondria, which in turn stimulates the production of reactive oxygen species. Taken together, the findings demonstrate that MIF-directed small molecules enhance ferroptosis via a putative MIF-BRCA1-RAD51 axis in HR, which causes resistance to ferroptosis. This suggests a potential novel druggable route to enhance ferroptosis by targeted anticancer therapeutics in the future.
PubMed: 38924362
DOI: 10.1002/advs.202403963