RESUMEN

Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

Asunto(s)

Inestabilidad Genómica , Neoplasias Ováricas , Ftalazinas , Piperazinas , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Femenino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Humanos , Animales , Línea Celular Tumoral , Ratones , Inestabilidad Genómica/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Autoantígeno Ku/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos

RESUMEN

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Asunto(s)

Daño del ADN , Proteína Quinasa Activada por ADN , Estrés del Retículo Endoplásmico , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteína Quinasa Activada por ADN/genética , Retículo Endoplásmico/metabolismo , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/genética , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación

RESUMEN

Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.

Asunto(s)

Antígeno B7-H1 , Núcleo Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Autoantígeno Ku , Humanos , Roturas del ADN de Doble Cadena/efectos de la radiación , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/patología , Ratones , Glicosilación , Radiación Ionizante , Sistemas CRISPR-Cas

RESUMEN

The Ku heterodimer (Ku70/Ku80) is central to the non-homologous end-joining (NHEJ) pathway. Ku binds to the broken DNA ends and promotes the assembly of the DNA repair complex. The N-terminal Ku70 von Willebrand A (vWA) domain is known to mediate protein-protein interactions important for the repair process. In particular, the D192 and D195 residues within helix 5 of the Ku70 vWA domain were shown to be essential for NHEJ function, although the precise role of these residues was not identified. Here, we set up a miniTurbo screening system to identify Ku70 D192/D195 residue-specific interactors in a conditional, human Ku70-knockout cell line in response to DNA damage. Using fusion protein constructs of Ku70 wild-type and mutant (D192A/D195R) with miniTurbo, we identified a number of candidate proximal interactors in response to DNA damage treatment, including DNA Ligase IV (LigIV), a known and essential NHEJ complex member. Interestingly, LigIV was enriched in our wildtype screen but not the Ku70 D192A/D195R screen, suggesting its interaction is disrupted by the mutation. Validation experiments demonstrated that the DNA damage-induced interaction between Ku70 and LigIV was disrupted by the Ku70 D192A/D195R mutations. Our findings provide greater detail about the interaction surface between the Ku70 vWA domain and LigIV and offer strong evidence that the D192 and D195 residues are important for NHEJ completion through an interaction with LigIV. Altogether, this work reveals novel potential proximal interactors of Ku in response to DNA damage and identifies Ku70 D192/D195 residues as essential for LigIV interaction with Ku during NHEJ.

Asunto(s)

Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP) , Proteínas de Unión al ADN , Autoantígeno Ku , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Humanos , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/metabolismo , ADN Ligasa (ATP)/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Daño del ADN , Unión Proteica , Dominios Proteicos , Factor de von Willebrand/metabolismo , Factor de von Willebrand/genética , Factor de von Willebrand/química

RESUMEN

Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.

Asunto(s)

Proteínas Bacterianas , Reparación del ADN por Unión de Extremidades , Autoantígeno Ku , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Autoantígeno Ku/metabolismo , Autoantígeno Ku/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Simulación de Dinámica Molecular , Unión Proteica , Simulación por Computador

RESUMEN

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements.

Asunto(s)

Reparación del ADN por Unión de Extremidades , Proteína Quinasa Activada por ADN , Primates , Animales , Proteína Quinasa Activada por ADN/metabolismo , Proteína Quinasa Activada por ADN/genética , Humanos , Primates/genética , Primates/metabolismo , Roturas del ADN de Doble Cadena , ARN/metabolismo , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Evolución Molecular , Mamíferos/metabolismo , Mamíferos/genética

RESUMEN

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

Asunto(s)

Cromatina , Proteína Quinasa Activada por ADN , Autoantígeno Ku , Cromatina/metabolismo , Autoantígeno Ku/metabolismo , Humanos , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo

RESUMEN

BACKGROUND: LINC-PINT was downregulated in nasopharyngeal carcinoma (NPC) and correlated with treatment efficiency of NPC. However, the underlying mechanism of LINC-PINT in NPC has not yet been fully explored. METHOD: We used CellTiter luminescent assay, clone formation assay, Hoechst staining, and SYTO-9/PI staining to examine cell viability and cell apoptosis regulated by LINC-PINT in NPC cells. Xenograft tumor model, HE staining, Ki67 staining, and TUNEL assay were conducted to assess the role of LINC-PINT in vivo. Bioinformatics and RNA immunoprecipitation assay was performed to identify the binding protein of LINC-PINT. Fluorescence in situ hybridization and immunofluorescence were utilized to measure the colocalization of XRCC6 with LINC-PINT and DNA-PKcs. Mito-Tracker red CMXRos staining was used to label mitochondria in cells specifically. RESULT: We found LINC-PINT was downregulated in many tumors (including NPC) and associated with poor prognosis. The cell viability was significantly inhibited and cell apoptosis was remarkably promoted in LINC-PINT overexpressed cells in contrast to control cells. The growth of tumor xenografts was significantly suppressed and the tumor weight was significantly decreased in LINC-PINT overexpression group compared to the control group. Correspondingly, the positive Ki67 foci was decreased while TUNEL foci was increased in LINC-PINT overexpression group. Mechanically, we verified XRCC6 as a new binding protein of LINC-PINT through RNA binding domains prediction, RIP and colocalization of LINC-PINT and XRCC6. By binding to XRCC6, LINC-PINT interfered the formation of DNA-PK complex, regulated mitochondria accumulation status and affected the modification of apoptosis proteins, leading to more cell apoptosis. CONCLUSION: Our study provided the first evidence that LINC-PINT promotes cell apoptosis in NPC by binding to XRCC6 and affecting its function.

Asunto(s)

Apoptosis , Autoantígeno Ku , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , ARN Largo no Codificante , Humanos , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/genética , Autoantígeno Ku/metabolismo , Ratones , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Ratones Desnudos , Línea Celular Tumoral

RESUMEN

Myositis with anti-Ku-autoantibodies is a rare inflammatory myopathy associated with various connective tissue diseases. Histopathological studies have identified inflammatory and necrotizing aspects, but a precise morphological analysis and pathomechanistic disease model are lacking. We therefore aimed to carry out an in-depth morpho-molecular analysis to uncover possible pathomechanisms. Muscle biopsy specimens from 26 patients with anti-Ku-antibodies and unequivocal myositis were analyzed by immunohistochemistry, immunofluorescence, transcriptomics, and proteomics and compared to biopsy specimens of non-disease controls, immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Clinical findings and laboratory parameters were evaluated retrospectively and correlated with morphological and molecular features. Patients were mainly female (92%) with a median age of 56.5 years. Isolated myositis and overlap with systemic sclerosis were reported in 31%, respectively. Isolated myositis presented with higher creatine kinase levels and cardiac involvement (83%), whereas systemic sclerosis-overlap patients often had interstitial lung disease (57%). Histopathology showed a wide spectrum from mild to pronounced myositis with diffuse sarcolemmal MHC-class I (100%) and -II (69%) immunoreactivity, myofiber necrosis (88%), endomysial inflammation (85%), thickened capillaries (84%), and vacuoles (60%). Conspicuous sarcoplasmic protein aggregates were p62, BAG3, myotilin, or immunoproteasomal beta5i-positive. Proteomic and transcriptomic analysis identified prominent up-regulation of autophagy, proteasome, and hnRNP-related cell stress. To conclude, Ku + myositis is morphologically characterized by myofiber necrosis, MHC-class I and II positivity, variable endomysial inflammation, and distinct protein aggregation varying from IBM and IMNM, and it can be placed in the spectrum of scleromyositis and overlap myositis. It features characteristic sarcoplasmic protein aggregation on an acquired basis being functionally associated with altered chaperone, proteasome, and autophagy function indicating that Ku + myositis exhibit aspects of an acquired inflammatory protein-aggregate myopathy.

Asunto(s)

Autoanticuerpos , Autoantígeno Ku , Miositis , Humanos , Femenino , Persona de Mediana Edad , Masculino , Miositis/patología , Miositis/inmunología , Miositis/metabolismo , Anciano , Autoanticuerpos/inmunología , Adulto , Autoantígeno Ku/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/metabolismo , Estudios Retrospectivos , Miositis por Cuerpos de Inclusión/patología , Miositis por Cuerpos de Inclusión/metabolismo

RESUMEN

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

Asunto(s)

Proteína BRCA1 , Neoplasias Colorrectales , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , ADN Polimerasa theta , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animales

RESUMEN

PURPOSE: Ovarian aging is closely related to a decrease in follicular reserve and oocyte quality. The precise molecular mechanisms underlying these reductions have yet to be fully elucidated. Herein, we examine spatiotemporal distribution of key proteins responsible for DNA double-strand break (DSB) repair in ovaries from early to older ages. Functional studies have shown that the γH2AX, RAD51, BRCA1, and RPA70 proteins play indispensable roles in HR-based repair pathway, while the KU80 and XRCC4 proteins are essential for successfully operating cNHEJ pathway. METHODS: Female Balb/C mice were divided into five groups as follows: Prepuberty (3 weeks old; n = 6), puberty (7 weeks old; n = 7), postpuberty (18 weeks old; n = 7), early aged (52 weeks old; n = 7), and late aged (60 weeks old; n = 7). The expression of DSB repair proteins, cellular senescence (ß-GAL) and apoptosis (cCASP3) markers was evaluated in the ovaries using immunohistochemistry. RESULT: ß-GAL and cCASP3 levels progressively increased from prepuberty to aged groups (P < 0.05). Notably, γH2AX levels varied in preantral and antral follicles among the groups (P < 0.05). In aged groups, RAD51, BRCA1, KU80, and XRCC4 levels increased (P < 0.05), while RPA70 levels decreased (P < 0.05) compared to the other groups. CONCLUSIONS: The observed alterations were primarily attributed to altered expression in oocytes and granulosa cells of the follicles and other ovarian cells. As a result, the findings indicate that these DSB repair proteins may play a role in the repair processes and even other related cellular events in ovarian cells from early to older ages.

Asunto(s)

Proteína BRCA1 , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN , Histonas , Autoantígeno Ku , Folículo Ovárico , Ovario , Recombinasa Rad51 , Animales , Femenino , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Ratones , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Reparación del ADN/genética , Folículo Ovárico/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Histonas/genética , Histonas/metabolismo , Ovario/metabolismo , Ovario/crecimiento & desarrollo , Oocitos/metabolismo , Oocitos/crecimiento & desarrollo , Envejecimiento/genética , Envejecimiento/metabolismo , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Ratones Endogámicos BALB C

RESUMEN

BACKGROUND: The escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC. METHODS: The key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance-related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay. RESULTS: NASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC. CONCLUSIONS: The reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

Asunto(s)

Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Autoantígeno Ku , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Oxaliplatino , ARN Largo no Codificante , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Ratones , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Animales , ARN Largo no Codificante/genética , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patología , Masculino , Femenino , Línea Celular Tumoral , Ratones Desnudos , Persona de Mediana Edad , Ensayos Antitumor por Modelo de Xenoinjerto , Adenosina/análogos & derivados

RESUMEN

Yes-associated protein (YAP) is a central player in cancer development, with functions extending beyond its recognized role in cell growth regulation. Recent work has identified a link between YAP/transcriptional coactivator with PDZ-binding motif (TAZ) and the DNA damage response. Here, we investigated the mechanistic underpinnings of the cross-talk between DNA damage repair and YAP activity. Ku70, a key component of the nonhomologous end joining pathway to repair DNA damage, engaged in a dynamic competition with TEAD4 for binding to YAP, limiting the transcriptional activity of YAP. Depletion of Ku70 enhanced interaction between YAP and TEAD4 and boosted YAP transcriptional capacity. Consequently, Ku70 loss enhanced tumorigenesis in colon cancer and hepatocellular carcinoma (HCC) in vivo. YAP impeded DNA damage repair and elevated genome instability by inducing PARP1 degradation through the SMURF2-mediated ubiquitin-proteasome pathway. Analysis of samples from patients with HCC substantiated the link between Ku70 expression, YAP activity, PARP1 levels, and genome instability. In conclusion, this research provides insight into the mechanistic interactions between YAP and key regulators of DNA damage repair, highlighting the role of a Ku70-YAP-PARP1 axis in preserving genome stability. Significance: Increased yes-associated protein transcriptional activity stimulated by loss of Ku70 induces PARP1 degradation by upregulating SMURF2 to inhibit DNA damage, driving genome instability and tumorigenesis.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas de Unión al ADN , Inestabilidad Genómica , Autoantígeno Ku , Poli(ADP-Ribosa) Polimerasa-1 , Factores de Transcripción , Ubiquitinación , Proteínas Señalizadoras YAP , Humanos , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Señalizadoras YAP/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Animales , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Ratones , Carcinogénesis/genética , Carcinogénesis/metabolismo , Factores de Transcripción de Dominio TEA/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Daño del ADN , Reparación del ADN , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones Desnudos

RESUMEN

Genomic instability is one of the hallmarks of cancer. While loss of histone demethylase KDM6A increases the risk of tumorigenesis, its specific role in maintaining genomic stability remains poorly understood. Here, we propose a mechanism in which KDM6A maintains genomic stability independently on its demethylase activity. This occurs through its interaction with SND1, resulting in the establishment of a protective chromatin state that prevents replication fork collapse by recruiting of RPA and Ku70 to nascent DNA strand. Notably, KDM6A-SND1 interaction is up-regulated by KDM6A SUMOylation, while KDM6AK90A mutation almost abolish the interaction. Loss of KDM6A or SND1 leads to increased enrichment of H3K9ac and H4K8ac but attenuates the enrichment of Ku70 and H3K4me3 at nascent DNA strand. This subsequently results in enhanced cellular sensitivity to genotoxins and genomic instability. Consistent with these findings, knockdown of KDM6A and SND1 in esophageal squamous cell carcinoma (ESCC) cells increases genotoxin sensitivity. Intriguingly, KDM6A H101D & P110S, N1156T and D1216N mutations identified in ESCC patients promote genotoxin resistance via increased SND1 association. Our finding provides novel insights into the pivotal role of KDM6A-SND1 in genomic stability and chemoresistance, implying that targeting KDM6A and/or its interaction with SND1 may be a promising strategy to overcome the chemoresistance.

Asunto(s)

Resistencia a Antineoplásicos , Inestabilidad Genómica , Histona Demetilasas , Humanos , Inestabilidad Genómica/genética , Resistencia a Antineoplásicos/genética , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Línea Celular Tumoral , Mutación , Histonas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Sumoilación , Endonucleasas/metabolismo , Endonucleasas/genética , Replicación del ADN , Cromatina/metabolismo , Cromatina/genética , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética

RESUMEN

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the nonhomologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Asunto(s)

Glucolípidos , Recombinación Homóloga , Autoantígeno Ku , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Glucolípidos/biosíntesis , Glucolípidos/metabolismo , Ustilaginales/genética , Ustilaginales/metabolismo , Bleomicina/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Marcadores Genéticos , Eliminación de Gen

RESUMEN

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Histonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Humanos , Metilación , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Recombinación Homóloga , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Reparación del ADN , Cromatina/metabolismo , Cromatina/genética

RESUMEN

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

Asunto(s)

Cricetulus , Replicación del ADN , Autoantígeno Ku , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Animales , Células CHO , Humanos , Cricetinae , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Helicasa del Síndrome de Werner/metabolismo , Helicasa del Síndrome de Werner/genética , Reparación del ADN por Unión de Extremidades , Unión Proteica , Camptotecina/farmacología , Hidroxiurea/farmacología

RESUMEN

The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC50 value of 2.3 µM. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment.

Asunto(s)

Antineoplásicos , Autoantígeno Ku , Humanos , Autoantígeno Ku/metabolismo , Animales , Línea Celular Tumoral , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Daño del ADN/efectos de los fármacos , Simulación del Acoplamiento Molecular , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Etopósido/farmacología , Descubrimiento de Drogas , Roturas del ADN de Doble Cadena/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos

RESUMEN

BACKGROUND/AIM: Automated measurement of immunostained samples can enable more convenient and objective prediction of treatment outcome from radiotherapy. We aimed to validate the performance of the QuPath image analysis software in immune cell markers detection by comparing QuPath cell counting results with those of physician manual cell counting. PATIENTS AND METHODS: CD8- and FoxP3-stained cervical, CD8-stained oropharyngeal, and Ku70-stained prostate cancer tumor sections were analyzed in 104 cervical, 92 oropharyngeal, and 58 prostate cancer patients undergoing radiotherapy at our Institution. RESULTS: QuPath and manual counts were highly correlated. When divided into two groups using ROC curves, the agreement between QuPath and manual counts was 89.4% for CD8 and 88.5% for FoxP3 in cervical cancer, 87.0% for CD8 in oropharyngeal cancer and 80.7% for Ku70 in prostate cancer. In cervical cancer, the high CD8 group based on QuPath counts had a better prognosis and the low CD8 group had a significantly worse prognosis [p=0.0003; 5-year overall survival (OS), 65.9% vs. 34.7%]. QuPath counts were more predictive than manual counts. Similar results were observed for FoxP3 in cervical cancer (p=0.002; 5-year OS, 62.1% vs. 33.6%) and CD8 in oropharyngeal cancer (p=0.013; 5-year OS, 80.2% vs. 47.2%). In prostate cancer, high Ku70 group had worse and low group significantly better outcome [p=0.007; 10-year progression-free survival (PFS), 56.0% vs. 93.8%]. CONCLUSION: QuPath showed a strong correlation with manual counting, confirming its utility and accuracy and potential applicability in clinical practice.

Asunto(s)

Programas Informáticos , Humanos , Masculino , Femenino , Pronóstico , Persona de Mediana Edad , Anciano , Resultado del Tratamiento , Biomarcadores de Tumor/metabolismo , Adulto , Autoantígeno Ku/metabolismo , Factores de Transcripción Forkhead/metabolismo , Neoplasias de la Próstata/radioterapia , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Curva ROC , Antígenos CD8/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Neoplasias/radioterapia , Neoplasias/metabolismo , Neoplasias/patología

RESUMEN

The development of resistance to cisplatin (CDDP) in bladder cancer presents a notable obstacle, with indications pointing to the substantial role of circular RNAs (circRNAs) in this resistance. Nevertheless, the precise mechanisms through which circRNAs govern resistance are not yet fully understood. Our findings demonstrate that circUGGT2 is significantly upregulated in bladder cancer, facilitating cancer cell migration and invasion. Additionally, our analysis of eighty patient outcomes revealed a negative correlation between circUGGT2 expression levels and prognosis. Using circRNA pull-down assays, mass spectrometry analyses, and RNA Immunoprecipitation (RIP), it was shown that circUGGT2 interacts with the KU heterodimer, consisting of KU70 and KU80. Both KU70 and KU80 are critical components of the non-homologous end joining (NHEJ) pathway, which plays a role in CDDP resistance. Flow cytometry was utilized in this study to illustrate the impact of circUGGT2 on the sensitivity of bladder cancer cell lines to CDDP through its interaction with KU70 and KU80. Additionally, a reduction in the levels of DNA repair factors associated with the NHEJ pathway, such as KU70, KU80, DNA-PKcs, and XRCC4, was observed in chromatin of bladder cancer cells following circUGGT2 knockdown post-CDDP treatment, while the levels of DNA repair factors in total cellular proteins remained constant. Thus, the promotion of CDDP resistance by circUGGT2 is attributed to its facilitation of repair factor recruitment to DNA breaks via interaction with the KU heterodimer. Furthermore, our study demonstrated that knockdown of circUGGT2 resulted in reduced levels of γH2AX, a marker of DNA damage response, in CDDP-treated bladder cancer cells, implicating circUGGT2 in the NHEJ pathway for DNA repair.

Asunto(s)

Cisplatino , Reparación del ADN por Unión de Extremidades , Resistencia a Antineoplásicos , Autoantígeno Ku , ARN Circular , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , ARN Circular/metabolismo , ARN Circular/genética , Línea Celular Tumoral , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad