RESUMEN
Resistance to olaparib is the major obstacle in targeted therapy for ovarian cancer (OC) with poly(ADP-ribose) polymerase inhibitors (PARPis), prompting studies on novel combination therapies to enhance olaparib efficacy. Despite identifying various mechanisms, understanding how OC cells acquire PARPi resistance remains incomplete. This study investigated microRNA (miRNA) expression in olaparib-sensitive (PEO1, PEO4) and previously established olaparib-resistant OC cell lines (PEO1-OR) using high-throughput RT-qPCR and bioinformatic analyses. The role of miRNAs was explored regarding acquired resistance and resensitization with the ATR/CHK1 pathway inhibitors. Differentially expressed miRNAs were used to construct miRNA-mRNA regulatory networks and perform functional enrichment analyses for target genes with miRNet 2.0. TCGA-OV dataset was analyzed to explore the prognostic value of selected miRNAs and target genes in clinical samples. We identified potential processes associated with olaparib resistance, including cell proliferation, migration, cell cycle, and growth factor signaling. Resensitized PEO1-OR cells were enriched in growth factor signaling via PDGF, EGFR, FGFR1, VEGFR2, and TGFßR, regulation of the cell cycle via the G2/M checkpoint, and caspase-mediated apoptosis. Antibody microarray analysis confirmed dysregulated growth factor expression. The addition of the ATR/CHK1 pathway inhibitors to olaparib downregulated FGF4, FGF6, NT-4, PLGF, and TGFß1 exclusively in PEO1-OR cells. Survival and differential expression analyses for serous OC patients revealed prognostic miRNAs likely associated with olaparib resistance (miR-99b-5p, miR-424-3p, and miR-505-5p) and resensitization to olaparib (miR-324-5p and miR-424-3p). Essential miRNA-mRNA interactions were reconstructed based on prognostic miRNAs and target genes. In conclusion, our data highlight distinct miRNA profiles in olaparib-sensitive and olaparib-resistant cells, offering molecular insights into overcoming resistance with the ATR/CHK1 inhibitors in OC. Moreover, some miRNAs might serve as potential predictive signature molecules of resistance and therapeutic response.
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Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA2 , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs , Neoplasias Ováricas , Ftalazinas , Piperazinas , ARN Mensajero , Humanos , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , MicroARNs/genética , MicroARNs/metabolismo , Femenino , Piperazinas/farmacología , Piperazinas/uso terapéutico , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Línea Celular Tumoral , Redes Reguladoras de Genes/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Proteínas de Unión al ADN , Humanos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Fosforilación , Proteínas de Unión al ADN/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Proteínas Portadoras/metabolismo , Replicación del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteína BRCA1/metabolismo , Transducción de Señal , Proteínas Nucleares/metabolismo , Fibroblastos/metabolismo , Puntos de Control del Ciclo CelularRESUMEN
The prevalence of oral squamous cell carcinoma (OSCC) is increasing worldwide mainly due to poor oral hygiene and unrestricted lifestyle. Advanced-stage OSCC is associated with poor prognosis and a 5-year survival rate of only 30%-50%. The present study was designed to investigate the anticancer effect and mode of action of Glycyrrhiza-derived semilicoisoflavone B (SFB) in 5-fluorourasil (5FU)-resistant human OSCC cell lines. The study findings revealed that SFB significantly reduces OSCC cell viability and colony formation ability by arresting cell cycle at the G2/M and S phases and reducing the expressions of key cell cycle regulators including cyclin A, cyclin B, CDC2, and CDK2. The compound caused a significant induction in the percentage of nuclear condensation and apoptotic cells in OSCC. Regarding pro-apoptotic mode of action, SFB was found to increase Fas-associated death domain and death receptor 5 expressions and reduce decoy receptor 2 expression, indicating involvement of extrinsic pathway. Moreover, SFB was found to increase pro-apoptotic Bim expression and reduce anti-apoptotic Bcl-2 and Bcl-xL expressions, indicating involvement of intrinsic pathway. Moreover, SFB-mediated induction in cleaved caspases 3, 8, and 9 and cleaved poly(ADP-ribose) polymerase confirmed the induction of caspase-mediated apoptotic pathways. Regarding upstream signaling pathway, SFB was found to reduce extracellular signal regulated kinase 1/2 (ERK) phosphorylation to execute its pro-apoptotic activity. The Human Apoptotic Array findings revealed that SFB suppresses claspin expression, which in turn caused reduced phosphorylation of ATR, checkpoint kinase 1 (Chk1), Wee1, and CDC25C, indicating disruption of ATR-Chk1 signaling pathway by SFB. Taken together, these findings indicate that SFB acts as a potent anticancer compound against 5FU-resistant OSCC by modulating mitogen-activated protein kinase (MAPK) and ATR-Chk1 signaling pathways.
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Carcinoma de Células Escamosas , Flavonoides , Neoplasias de la Boca , Humanos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Apoptosis , Transducción de Señal , Fosforilación , Fluorouracilo , Línea Celular Tumoral , Proteínas de la Ataxia Telangiectasia MutadaRESUMEN
DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells.
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Reparación del ADN , Replicación del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , ADN , Daño del ADN , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismoRESUMEN
Endometrial cancer (EC) is increasingly undermining female health worldwide, with poor survival rates for advanced or recurrent/metastatic diseases. The application of immune checkpoint inhibitors (ICIs) has opened a window of opportunity for patients with first-line therapy failure. However, there is a subset of patients with endometrial cancer who remain insensitive to immunotherapy alone. Therefore, it is necessary to develop new therapeutic agents and further explore reliable combinational strategies to optimize the efficacy of immunotherapy. DNA damage repair (DDR) inhibitors as novel targeted drugs are able to generate genomic toxicity and induce cell death in solid tumors, including EC. Recently, growing evidence has demonstrated the DDR pathway modulates innate and adaptive immunity in tumors. In this review, we concentrate on the exploration of the intrinsic correlation between DDR pathways, especially the ATM-CHK2-P53 pathway and the ATR-CHK1-WEE1 pathway, and oncologic immune response, as well as the feasibility of adding DDR inhibitors to ICIs for the treatment of patients with advanced or recurrent/metastatic EC. We hope that this review will offer some beneficial references to the investigation of immunotherapy and provide a reasonable basis for "double-checkpoint inhibition" in EC.
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The PARP inhibitor (PARPi) olaparib is currently the drug of choice for serous ovarian cancer (OC), especially in patients with homologous recombination (HR) repair deficiency associated with deleterious BRCA1/2 mutations. Unfortunately, OC patients who fail to respond to PARPi or relapse after treatment have limited therapeutic options. To elucidate olaparib resistance and enhance the efficacy of olaparib, intracellular factors exploited by OC cells to achieve decreased sensitivity to PARPi were examined. An olaparib-resistant OC cell line, PEO1-OR, was established from BRCA2MUT PEO1 cells. The anticancer activity and action of olaparib combined with inhibitors of the ATR/CHK1 pathway (ceralasertib as ATRi, MK-8776 as CHK1i) in olaparib-sensitive and -resistant OC cell lines were evaluated. Whole-exome sequencing revealed that PEO1-OR cells acquire resistance through subclonal enrichment of BRCA2 secondary mutations that restore functional full-length protein. Moreover, PEO1-OR cells upregulate HR repair-promoting factors (BRCA1, BRCA2, RAD51) and PARP1. Olaparib-inducible activation of the ATR/CHK1 pathway and G2/M arrest is abrogated in olaparib-resistant cells. Drug sensitivity assays revealed that PEO1-OR cells are less sensitive to ATRi and CHK1i agents. Combined treatment is less effective in olaparib-resistant cells considering inhibition of metabolic activity, colony formation, survival, accumulation of DNA double-strand breaks, and chromosomal aberrations. However, synergistic antitumor activity between compounds is achievable in PEO1-OR cells. Collectively, olaparib-resistant cells display co-existing HR repair-related mechanisms that confer resistance to olaparib, which may be effectively utilized to resensitize them to PARPi via combination therapy. Importantly, the addition of ATR/CHK1 pathway inhibitors to olaparib has the potential to overcome acquired resistance to PARPi.
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Antineoplásicos , Neoplasias Ováricas , Humanos , Femenino , Apoptosis , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Daño del ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína BRCA2/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
Lung cancer is the leading cause of cancer-related death. In particular, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Due to tumor resistance and the toxicity of chemotherapeutic agents, it is increasingly critical to discover novel, potent antitumorigenic drugs for treating NSCLC. Lutein, a carotenoid, has been reported to exert toxic effects on cells in several tumor types. However, the detailed functions and underlying mechanisms of lutein in NSCLC remain elusive. The present study showed that lutein significantly and dose-dependently inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase, and induced apoptosis in NSCLC cells. RNA-sequencing analysis revealed that the p53 signaling pathway was the most significantly upregulated in lutein-treated A549 cells. Mechanistically, lutein exerted antitumorigenic effects by inducing DNA damage and subsequently activating the ATR/Chk1/p53 signaling pathway in A549 cells. In vivo, lutein impeded tumor growth in mice and prolonged their survival. In conclusion, our findings demonstrate the antitumorigenic potential of lutein and reveal its molecular mechanism of action, suggesting that lutein is a promising candidate for clinical NSCLC treatment.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Luteína/metabolismo , Luteína/farmacología , Luteína/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Transducción de SeñalRESUMEN
Accurate replication of the genome is fundamental to cellular survival and tumor prevention. The DNA replication fork is vulnerable to DNA lesions and damages that impair replisome progression, and improper control over DNA replication stress inevitably causes fork stalling and collapse, a major source of genome instability that fuels tumorigenesis. The integrity of the DNA replication fork is maintained by the fork protection complex (FPC), in which TIMELESS (TIM) constitutes a key scaffold that couples the CMG helicase and replicative polymerase activities, in conjunction with its interaction with other proteins associated with the replication machinery. Loss of TIM or the FPC in general results in impaired fork progression, elevated fork stalling and breakage, and a defect in replication checkpoint activation, thus underscoring its pivotal role in protecting the integrity of both active and stalled replication forks. TIM is upregulated in multiple cancers, which may represent a replication vulnerability of cancer cells that could be exploited for new therapies. Here, we discuss recent advances on our understanding of the multifaceted roles of TIM in DNA replication and stalled fork protection, and how its complex functions are engaged in collaboration with other genome surveillance and maintenance factors.
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Replicación del ADN , ADN , ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN Helicasas/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
DNA replication forks are frequently forced into stalling by persistent DNA aberrations generated from endogenous or exogenous insults. Stalled replication forks are catastrophic for genome integrity and cell survival if not immediately stabilized. The ataxia-telangiectasia and RAD3-related kinase (ATR)-CLASPIN-checkpoint kinase 1 (CHK1) signaling cascade is a pivotal mechanism that initiates cell-cycle checkpoints and stabilizes stalled replication forks, assuring the faithful duplication of genomic information before entry into mitosis. The timely recovery of checkpoints after stressors are resolved is also crucial for normal cell proliferation. The precise activation and inactivation of ATR-CHK1 signaling are usually efficiently regulated by turnover and the cellular re-localization of the adaptor protein CLASPIN. The ubiquitination-proteasome-mediated degradation of CLASPIN, driven by APC/CCDH1 and SCFßTrCP, results in a cell-cycle-dependent fluctuation pattern of CLASPIN levels, with peak levels seen in S/G2 phase when it functions in the DNA replisome or as an adaptor protein in ATR-CHK1 signaling under replication stress. Deubiquitination mediated by a series of ubiquitin-specific protease family proteins releases CLASPIN from proteasome-dependent destruction and activates the ATR-CHK1 checkpoint to overcome replication stress. Moreover, the non-proteolytic ubiquitination of CLASPIN also affects CHK1 activation by regulating CLASPIN localization. In this review, we discuss the functions of CLASPIN ubiquitination with specific linkage types in the regulation of the ATR-CHK1 signaling pathway. Research in this area is progressing at pace and provides promising chemotherapeutic targets.
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Replicación del ADN , Complejo de la Endopetidasa Proteasomal , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitinación , Proteínas de Ciclo Celular/metabolismo , Transducción de Señal , Fosforilación , Daño del ADNRESUMEN
Cancer is one of the most severe medical conditions in the world, causing millions of deaths each year. Chemotherapy and radiotherapy are critical for treatment approaches, but both have numerous adverse health effects. Furthermore, the resistance of cancerous cells to anticancer medication leads to treatment failure. The rising burden of cancer requires novel efficacious treatment modalities. Natural remedies offer feasible alternative options against malignancy in contrast to available synthetic medication. Selective killing of cancer cells is privileged mainstream in cancer treatment, and targeted therapy represents the new tool with the potential to pursue this aim. The discovery of innovative therapies targeting essential components of DNA damage signaling and repair pathways such as ataxia telangiectasia mutated and Rad3 related Checkpoint kinase 1 (ATR-CHK1)has offered a possibility of significant therapeutic improvement in oncology. The activation and inhibition of this pathway account for chemopreventive and chemotherapeutic activity, respectively. Targeting this pathway can also aid to overcome the resistance of conventional chemo- or radiotherapy. This review enlightens the anticancer role of natural products by ATR-CHK1 activation and inhibition. Additionally, these compounds have been shown to have chemotherapeutic synergistic potential when used in combination with other anticancer drugs. Ideally, this review will trigger interest in natural products targeting ATR-CHK1 and their potential efficacy and safety as cancer lessening agents.
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Productos Biológicos , Neoplasias , Humanos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Daño del ADN , Transducción de Señal , Neoplasias/tratamiento farmacológicoRESUMEN
As our understanding of the mechanisms of cancer treatment has increased, a growing number of studies demonstrate pathways through which DNA damage repair (DDR) affects the immune system. At the same time, the varied response of patients to immune checkpoint blockade (ICB) therapy has prompted the discovery of various predictive biomarkers and the study of combination therapy. Here, our investigation explores the interactions involved in combination therapy, accompanied by a review that summarizes currently identified and promising predictors of response to immune checkpoint inhibitors (ICIs) that are useful for classifying oncology patients. In addition, this work, which discusses immunogenicity and several components of the tumor immune microenvironment, serves to illustrate the mechanism by which higher response rates and improved efficacy of DDR inhibitors (DDRi) in combination with ICIs are achieved.
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Inhibidores de Puntos de Control Inmunológico , Neoplasias , Daño del ADN , Reparación del ADN , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Breast cancer and gynecological tumors seriously endanger women's physical and mental health, fertility, and quality of life. Due to standardized surgical treatment, chemotherapy, and radiotherapy, the prognosis and overall survival of cancer patients have improved compared to earlier, but the management of advanced disease still faces great challenges. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have been clinically approved for breast and gynecological cancer patients, significantly improving their quality of life, especially of patients with BRCA1/2 mutations. However, drug resistance faced by PARPi therapy has hindered its clinical promotion. Therefore, developing new drug strategies to resensitize cancers affecting women to PARPi therapy is the direction of our future research. Currently, the effects of PARPi in combination with other drugs to overcome drug resistance are being studied. In this article, we review the mechanisms of PARPi resistance and summarize the current combination of clinical trials that can improve its resistance, with a view to identify the best clinical treatment to save the lives of patients.
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ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.
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Proteínas de la Ataxia Telangiectasia Mutada , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN , Proteínas del Tejido Nervioso , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Cromatina/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Ratones , Microcefalia/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/metabolismoRESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have become a mainstay of therapy in ovarian cancer and other malignancies, including BRCA-mutant breast, prostate, and pancreatic cancers. However, a growing number of patients develop resistance to PARPis, highlighting the need to further understand the mechanisms of PARPi resistance and develop effective treatment strategies. Targeting cell cycle checkpoint protein kinases, e.g., ATR, CHK1, and WEE1, which are upregulated in response to replication stress, represents one such therapeutic approach for PARPi-resistant cancers. Mechanistically, activated cell cycle checkpoints promote cell cycle arrest, replication fork stabilization, and DNA repair, demonstrating the interplay of DNA repair proteins with replication stress in the development of PARPi resistance. Inhibitors of these cell cycle checkpoints are under investigation in PARPi-resistant ovarian and other cancers. In this review, we discuss the cell cycle checkpoints and their roles beyond mere cell cycle regulation as part of the arsenal to overcome PARPi-resistant cancers. We also address the current status and recent advancements as well as limitations of cell cycle checkpoint inhibitors in clinical trials.
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Antineoplásicos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Neoplasias Ováricas , Proteínas Tirosina Quinasas , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Actin-like 6A (ACTL6A) is a core subunit of the SWI/SNF chromatin remodeling complex and is highly expressed in several types of human cancers including glioblastoma. Recent studies verified that ACTL6A regulates the proliferation, differentiation, and migration of cancer cells. In this study, we identified ACTL6A as an important regulator of DNA replication. ACTL6A knockdown could impair the DNA replication initiation in glioblastoma cells. The regulation of DNA replication by ACTL6A was mediated through regulating the expression of the CDC45-MCM-GINS (CMG) complex genes. Further investigation revealed that ACTL6A transcriptionally regulates MCM5 expression. Furthermore, ACTL6A knockdown induced DNA damage and diminished the activity of the ATR-Chk1 pathway, which ultimately led glioblastoma cells to apoptosis and death. Taken together, our findings highlight the critical role of ACTL6A in DNA replication and ATR-Chk1 pathway, and reveal a potential target for therapeutic intervention in glioblastoma.
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Actinas/genética , Apoptosis/genética , Proteínas Cromosómicas no Histona/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Glioblastoma/genética , Glioblastoma/patología , Actinas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal/fisiologíaRESUMEN
Almost 50% myelodysplastic syndromes (MDS) patients have different splicing factor mutations, including SF3B1, SRSF2, U2AF1. Different splicing factor mutations cause the various mechanisms of slicing abnormality and eventually lead to the similar MDS phenotypes, indicating that splicing factor mutations might generate the common pathopoiesia pathway different from slicing abnormality. Recent studies have shown that SF3B1, U2AF1 and SRSF2 mutations could contribute to the accumulation of R-loop, cause DNA damage and repair abnormality, activate ATR-Chk1 pathway and finally promote apoptosis and tumorigenesis. This paper reviews the role of R-loop in the pathogenesis of MDS and the progress of related targeted drugs.
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The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3'-5' exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.
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Periodontitis is closely related to oral cancer, but the molecular mechanism of periodontal pathogens involved in the occurrence and development of oral cancer is still inconclusive. Here, we demonstrate that, in vitro, the cell proliferation ability and S phase cells of the periodontitis group (colonized by Porphyromonas gingivalis and Fusobacterium nucleatum, P+) significantly increased, but the G1 cells were obviously reduced. The animal models with an in situ oral squamous cell carcinoma (OSCC) and periodontitis-associated bacteria treatment were constructed, and micro-CT showed that the alveolar bone resorption of mice in the P+ group (75.3 ± 4.0 µm) increased by about 53% compared with that in the control group (48.8 ± 1.3 µm). The tumor mass and tumor growth rate in the P+ group were all higher than those in the blank control group. Hematoxylin-eosin (H&E) staining of isolated tumor tissues showed that large-scale flaky necrosis was found in the tumor tissue of the P+ group, with lots of damaged vascular profile and cell debris. Immunohistochemistry (IHC) of isolated tumor tissues showed that the expression of Ki67 and the positive rate of cyclin D1 were significantly higher in tumor tissues of the P+ group. The qRT-PCR results of the expression of inflammatory cytokines in oral cancer showed that periodontitis-associated bacteria significantly upregulated interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-18, apoptosis-associated speck-like protein containing a CARD (ASC) (up to six times), and caspase-1 (up to four times), but it downregulated nuclear factor (NF)-κB, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and IL-1ß (less than 0.5 times). In addition, the volume of spleen tissue and the number of CD4+ T cells, CD8+ T cells, and CD206+ macrophages in the P+ group increased significantly. IHC and Western blotting in tumor tissues showed that expression levels of γ-H2AX, p-ATR, RPA32, CHK1, and RAD51 were upregulated, and the phosphorylation level of CHK1 (p-chk1) was downregulated. Together, we identify that the periodontitis-related bacteria could promote tumor growth and proliferation, initiate the overexpressed NLRP3, and activate upstream signal molecules of ATR-CHK1. It is expected to develop a new molecular mechanism between periodontitis-related bacteria and OSCC.
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BACKGROUND: Liver cancer is one of the most commonly diagnosed cancers and the fourth leading cause of cancer-related death worldwide. Broad-spectrum kinase inhibitors like sorafenib and lenvatinib provide only modest survival benefit to patients with hepatocellular carcinoma (HCC). This study aims to identify novel therapeutic strategies for HCC patients. METHODS: Integrated bioinformatics analyses and a non-biased CRISPR loss of function genetic screen were performed to identify potential therapeutic targets for HCC cells. Whole-transcriptome sequencing (RNA-Seq) and time-lapse live imaging were performed to explore the mechanisms of the synergy between CDC7 inhibition and ATR or CHK1 inhibitors in HCC cells. Multiple in vitro and in vivo assays were used to validate the synergistic effects. RESULTS: Through integrated bioinformatics analyses using the Cancer Dependency Map and the TCGA database, we identified ATR-CHK1 signaling as a therapeutic target for liver cancer. Pharmacological inhibition of ATR or CHK1 leads to robust proliferation inhibition in liver cancer cells having a high basal level of replication stress. For liver cancer cells that are resistant to ATR or CHK1 inhibition, treatment with CDC7 inhibitors induces strong DNA replication stress and consequently such drugs show striking synergy with ATR or CHK1 inhibitors. The synergy between ATR-CHK1 inhibition and CDC7 inhibition probably derives from abnormalities in mitosis inducing mitotic catastrophe. CONCLUSIONS: Our data highlights the potential of targeting ATR-CHK1 signaling, either alone or in combination with CDC7 inhibition, for the treatment of liver cancer.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Replicación del ADN , Neoplasias Hepáticas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
APE2 is a rising vital player in the maintenance of genome and epigenome integrity. In the past several years, a series of studies have shown the critical roles and functions of APE2. We seek to provide the first comprehensive review on several aspects of APE2 in genome and epigenome integrity. We first summarize the distinct functional domains or motifs within APE2 including EEP (endonuclease/exonuclease/phosphatase) domain, PIP box and Zf-GRF motifs from eight species (i.e., Homo sapiens, Mus musculus, Xenopus laevis, Ciona intestinalis, Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Trypanosoma cruzi). Then we analyze various APE2 nuclease activities and associated DNA substrates, including AP endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-5' exonuclease activities. We also examine several APE2 interaction proteins, including PCNA, Chk1, APE1, Myh1, and homologous recombination (HR) factors such as Rad51, Rad52, BRCA1, BRCA2, and BARD1. Furthermore, we provide insights into the roles of APE2 in various DNA repair pathways (base excision repair, single-strand break repair, and double-strand break repair), DNA damage response (DDR) pathways (ATR-Chk1 and p53-dependent), immunoglobulin class switch recombination and somatic hypermutation, as well as active DNA demethylation. Lastly, we summarize critical functions of APE2 in growth, development, and diseases. In this review, we provide the first comprehensive perspective which dissects all aspects of the multiple-function protein APE2 in genome and epigenome integrity.