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Genes Jun 2024Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks...
Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks (DSBs) as cytotoxic DNA damage; thus, the DSB repair activity in each cancer cell significantly influences the efficacy of the treatments. Pancreatic cancers are known to be resistant to these treatments, and the overexpression of MUC1, a member of the glycoprotein mucins, is associated with IR- and chemo-resistance. Therefore, we investigated the impact of MUC1 on DSB repair. This report examined the effect of the overexpression of MUC1 on homologous recombination (HR) and non-homologous end-joining (NHEJ) using cell-based DSB repair assays. In addition, the therapeutic potential of NHEJ inhibitors including HDAC inhibitors was also studied using pancreatic cancer cell lines. The MUC1-overexpression enhances NHEJ, while partially suppressing HR. Also, MUC1-overexpressed cancer cell lines are preferentially killed by a DNA-PK inhibitor and HDAC1/2 inhibitors. Altogether, MUC1 induces metabolic changes that create an imbalance between NHEJ and HR activities, and this imbalance can be a target for selective killing by HDAC inhibitors. This is a novel mechanism of MUC1-mediated IR-resistance and will form the basis for targeting MUC1-overexpressed pancreatic cancer.
Topics: Humans; Mucin-1; DNA End-Joining Repair; Cell Line, Tumor; DNA Breaks, Double-Stranded; Pancreatic Neoplasms; Up-Regulation; Homologous Recombination; Histone Deacetylase Inhibitors; Gene Expression Regulation, Neoplastic
PubMed: 38927743
DOI: 10.3390/genes15060808 -
Genes Jun 2024Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG expansion on the huntingtin () gene and is characterized by progressive... (Review)
Review
Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG expansion on the huntingtin () gene and is characterized by progressive motor, cognitive, and neuropsychiatric decline. Recently, new genetic factors besides CAG repeats have been implicated in the disease pathogenesis. Most genetic modifiers are involved in DNA repair pathways and, as the cause of the loss of CAA interruption in the gene, they exert their main influence through somatic expansion. However, this mechanism might not be the only driver of HD pathogenesis, and future studies are warranted in this field. The aim of the present review is to dissect the many faces of genetics in HD pathogenesis, from cis- and trans-acting genetic modifiers to RNA toxicity, mitochondrial DNA mutations, and epigenetics factors. Exploring genetic modifiers of HD onset and progression appears crucial to elucidate not only disease pathogenesis, but also to improve disease prediction and prevention, develop biomarkers of disease progression and response to therapies, and recognize new therapeutic opportunities. Since the same genetic mechanisms are also described in other repeat expansion diseases, their implications might encompass the whole spectrum of these disorders.
Topics: Huntington Disease; Humans; Huntingtin Protein; Trinucleotide Repeat Expansion; Animals; Epigenesis, Genetic; DNA, Mitochondrial
PubMed: 38927742
DOI: 10.3390/genes15060807 -
Genes Jun 2024Deficiencies in DNA mismatch repair (MMRd) leave characteristic footprints of microsatellite instability (MSI) in cancer genomes. We used data from the Cancer Genome...
Deficiencies in DNA mismatch repair (MMRd) leave characteristic footprints of microsatellite instability (MSI) in cancer genomes. We used data from the Cancer Genome Atlas and International Cancer Genome Consortium to conduct a comprehensive analysis of MSI-associated cancers, focusing on indel mutational signatures. We classified MSI-high genomes into two subtypes based on their indel profiles: deletion-dominant (MMRd-del) and insertion-dominant (MMRd-ins). Compared with MMRd-del genomes, MMRd-ins genomes exhibit distinct mutational and transcriptomic features, including a higher prevalence of T>C substitutions and related mutation signatures. Short insertions and deletions in MMRd-ins and MMRd-del genomes target different sets of genes, resulting in distinct indel profiles between the two subtypes. In addition, indels in the MMRd-ins genomes are enriched with subclonal alterations that provide clues about a distinct evolutionary relationship between the MMRd-ins and MMRd-del genomes. Notably, the transcriptome analysis indicated that MMRd-ins cancers upregulate immune-related genes, show a high level of immune cell infiltration, and display an elevated neoantigen burden. The genomic and transcriptomic distinctions between the two types of MMRd genomes highlight the heterogeneity of genetic mechanisms and resulting genomic footprints and transcriptomic changes in cancers, which has potential clinical implications.
Topics: Humans; Microsatellite Instability; INDEL Mutation; Neoplasms; DNA Mismatch Repair; Genome, Human; Transcriptome
PubMed: 38927706
DOI: 10.3390/genes15060770 -
Genes Jun 2024Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after...
BACKGROUND
Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance.
MATERIALS AND METHODS
Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures.
RESULTS
Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ-test < 0.001).
CONCLUSION
Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.
Topics: Humans; Female; Drug Resistance, Neoplasm; Middle Aged; Ovarian Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors; Epigenesis, Genetic; Aged; DNA Methylation; Carcinoma, Ovarian Epithelial; Adult; Aneuploidy; Genomics
PubMed: 38927686
DOI: 10.3390/genes15060750 -
Biomedicines Jun 2024PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could...
PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could be pharmacologically impaired by the inhibition of histone lysine demethylases (KDM). Thus, we investigated whether KDM inhibitors could sensitize head and neck cancer cells, which are usually HR proficient, to PARP inhibition or cisplatin. Therefore, we explored the effects of double combinations of KDM4-6 inhibitors (ML324, CPI-455, GSK-J4, and JIB-04) with olaparib or cisplatin, or their triple combinations with both drugs, on the level of DNA damage and apoptosis. FaDu and SCC-040 cells were treated with individual compounds and their combinations, and cell viability, apoptosis, DNA damage, and gene expression were assessed using the resazurin assay, Annexin V staining, H2A.X activation, and qPCR, respectively. Combinations of KDM inhibitors with cisplatin enhanced cytotoxic effects, unlike combinations with olaparib. Triple combinations of KDM inhibitors with cisplatin and olaparib exhibited the best cytotoxic activity, which was associated with DNA damage accumulation and altered expression of genes associated with apoptosis induction and cell cycle arrest. In conclusion, triple combinations of KDM inhibitors (especially GSK-J4 and JIB-04) with cisplatin and olaparib represent a promising strategy for head and neck cancer treatment.
PubMed: 38927566
DOI: 10.3390/biomedicines12061359 -
Antibiotics (Basel, Switzerland) Jun 2024The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an...
The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against was 32-64 µg/mL, including strains carrying , which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene increased the MIC of 5-FU against by 32-fold, indicating the role of the gene in 5-FU resistance. Additionally, transcriptomic analysis of treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.
PubMed: 38927194
DOI: 10.3390/antibiotics13060528 -
Biomolecules Jun 2024Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks...
Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called "Christmas light models" to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.
Topics: Histones; DNA Breaks, Double-Stranded; Humans; Chromatin; Fluorescent Antibody Technique; Kinetics; Cell Nucleus; Fibroblasts; DNA Repair
PubMed: 38927105
DOI: 10.3390/biom14060703 -
Biomolecules Jun 2024Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for...
Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.
Topics: DNA Repair; Humans; Pyrimidine Nucleotides; Click Chemistry; DNA-Directed DNA Polymerase; Deoxyuridine; DNA; DNA Replication; Uracil-DNA Glycosidase
PubMed: 38927084
DOI: 10.3390/biom14060681 -
Journal of Ovarian Research Jun 2024Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of...
BACKGROUND
Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP.
METHODS
Human metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software.
RESULTS
We identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP.
CONCLUSIONS
Our findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest.
Topics: Humans; Oocytes; Female; Zona Pellucida; Transcriptome; Single-Cell Analysis; Sequence Analysis, RNA; Gene Expression Profiling; Adult
PubMed: 38926883
DOI: 10.1186/s13048-024-01463-8 -
Scientific Reports Jun 2024Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and...
Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, ERCC1, CENPX, and UBE2A were also dysregulated in siKHSRP samples and confirmed PCR experiments. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future.
Topics: Humans; Breast Neoplasms; Alternative Splicing; DNA Repair; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Female; Cell Proliferation; Cell Movement; RNA-Binding Proteins; Apoptosis; Carcinogenesis; MDA-MB-231 Cells
PubMed: 38926398
DOI: 10.1038/s41598-024-64687-0