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Developing novel therapies that outperform the existing chemotherapeutic treatments is required for treatment-resistant ovarian clear cell carcinoma. We investigated the antitumor effect of metformin on ovarian clear cell carcinoma, enhancement of the antitumor effect by its combination with chemotherapy, and its molecular regulatory mechanism. First, we evaluated the viability of ovarian clear cell carcinoma lines using the water-soluble tetrazolium-1 assay and found that metformin suppressed cell viability. Cell viability was significantly suppressed by co-treatment with cisplatin and metformin. In contrast, co-treatment with paclitaxel and metformin showed no significant difference in viability compared with the group without metformin. Western blot analysis showed increased phosphorylation of AMP-activated protein kinase in some cell lines and suppressed phosphorylation of the mammalian target of rapamycin in a particular cell line. Flow cytometry analysis revealed a significant increase in the rate of apoptosis in the metformin-treated group and rate of cell cycle arrest at the G2/M phase in a particular cell line. These results indicated that metformin may be effective against cultured ovarian clear cell carcinoma cells, particularly in combination with cisplatin. [ABSTRACT FROM AUTHOR]

Small nucleolar RNAs (snoRNAs) were previously regarded as a class of functionally conserved housekeeping genes, primarily involved in the regulation of ribosome biogenesis by ribosomal RNA (rRNA) modification. However, some of them are involved in several biological processes via complex molecular mechanisms. DNA damage response (DDR) is a conserved mechanism for maintaining genomic stability to prevent the occurrence of various human diseases. It has recently been revealed that snoRNAs are involved in DDR at multiple levels, indicating their relevant theoretical and clinical significance in this field. The present review systematically addresses four main points, including the biosynthesis and classification of snoRNAs, the mechanisms through which snoRNAs regulate target molecules, snoRNAs in the process of DDR, and the significance of snoRNA in disease diagnosis and treatment. It focuses on the potential functions of snoRNAs in DDR to help in the discovery of the roles of snoRNAs in maintaining genome stability and pathological processes. [ABSTRACT FROM AUTHOR]

Summary: Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage.Using a combination of whole‐mount single‐molecule RNA fluorescence in situ hybridization (WM‐smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots.The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases.This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants. [ABSTRACT FROM AUTHOR]

Abstract Small nucleolar RNAs (snoRNAs) were previously regarded as a class of functionally conserved housekeeping genes, primarily involved in the regulation of ribosome biogenesis by ribosomal RNA (rRNA) modification. However, some of them are involved in several biological processes via complex molecular mechanisms. DNA damage response (DDR) is a conserved mechanism for maintaining genomic stability to prevent the occurrence of various human diseases. It has recently been revealed that snoRNAs are involved in DDR at multiple levels, indicating their relevant theoretical and clinical significance in this field. The present review systematically addresses four main points, including the biosynthesis and classification of snoRNAs, the mechanisms through which snoRNAs regulate target molecules, snoRNAs in the process of DDR, and the significance of snoRNA in disease diagnosis and treatment. It focuses on the potential functions of snoRNAs in DDR to help in the discovery of the roles of snoRNAs in maintaining genome stability and pathological processes.

Simple Summary: Radium-223 is used for the management of metastatic castration-resistant prostate cancer. However, the response to this treatment is poor. Targeting the DNA damage response pathway through pharmacological inhibition has the ability to enhance the radiation response. In this study, we investigated the radiosensitising effects of three different DNA damage response inhibitors targeting ATM, ATR, and PARP in combination with X-rays and radium-223 on human prostate cancer cell lines. Their effects on cell survival, DNA double-strand break repair, cell cycle distribution, and apoptosis were evaluated. We determined that these inhibitors increase the ability of X-rays and radium-223 to kill prostate cells to varying degrees. Moreover, inhibition of the DNA damage response pathways impeded the repair of radiation-induced DNA double-strand breaks and induced changes in cell cycle distribution. Altogether, our study determined DDR inhibition as a promising strategy to increase the effectiveness of treatments utilising X-rays and/or 223Ra for localised and metastatic castration-resistant prostate cancer. Radium-223 (223Ra) and Lutetium-177-labelled-PSMA-617 (177Lu-PSMA) are currently the only radiopharmaceutical treatments to prolong survival for patients with metastatic-castration-resistant prostate cancer (mCRPC); however, mCRPC remains an aggressive disease. Recent clinical evidence suggests patients with mutations in DNA repair genes associated with homologous recombination have a greater clinical benefit from 223Ra. In this study, we aimed to determine the utility of combining DNA damage response (DDR) inhibitors to increase the therapeutic efficacy of X-rays, or 223Ra. Radiobiological responses were characterised by in vitro assessment of clonogenic survival, repair of double strand breaks, cell cycle distribution, and apoptosis via PARP-1 cleavage. Here, we show that DDR inhibitors increase the therapeutic efficacy of both radiation qualities examined, which is associated with greater levels of residual DNA damage. Co-treatment of ATM or PARP inhibition with 223Ra increased cell cycle arrest in the G2/M phase. In comparison, combined ATR inhibition and radiation qualities caused G2/M checkpoint abrogation. Additionally, greater levels of apoptosis were observed after the combination of DDR inhibitors with 223Ra. This study identified the ATR inhibitor as the most synergistic inhibitor for both radiation qualities, supporting further pre-clinical evaluation of DDR inhibitors in combination with 223Ra for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

A pulsed electromagnetic field (PEMF) enhances the efficacy of several anticancer drugs. Doxorubicin (DOX) is an anticancer agent used to treat various malignancies, including breast cancer. This study examined whether a PEMF increases the anticancer effect of DOX on MCF-7 human breast cancer cells and elucidated the underlying mechanisms affected by PEMF stimulation in DOX-treated MCF-7 human breast cancer cells. A cotreatment with DOX and a PEMF potentiated the reduction in MCF-7 cell viability compared to the treatment with DOX alone. The PEMF elevated DOX-induced G1 arrest by affecting cyclin-dependent kinase 2 phosphorylation and the expression of G1 arrest-related molecules, including p53, p21, cyclin E2, and polo like kinase 1. In addition, PEMF increased the DOX-induced upregulation of proapoptotic proteins, such as Fas and Bcl-2-associated X, and the downregulation of antiapoptotic proteins, including myeloid leukemia 1 and survivin. PEMF promoted the DOX-induced activation of caspases-8, -9, and -7 and poly (adenosine diphosphateribose) polymerase cleavage in MCF-7 cells. In conclusion, PEMF enhances the anticancer activity in DOX-treated MCF-7 breast cancer cells by increasing G1 cell cycle arrest and caspase-dependent apoptosis. These findings highlight the potential use of a PEMF as an adjuvant treatment for DOX-based chemotherapy against breast cancer.

Insulin resistance has been regarded as a hallmark of diabetes heart disease (DHD). Numerous studies have shown that insulin resistance can affect blood circulation and myocardium, which indirectly cause cardiac hypertrophy and ventricular remodeling, participating in the pathogenesis of DHD. Meanwhile, hyperinsulinemia, hyperglycemia, and hyperlipidemia associated with insulin resistance can directly impair the metabolism and function of the heart. Targeting insulin resistance is a potential therapeutic strategy for the prevention of DHD. Currently, the role of insulin resistance in the pathogenic development of DHD is still under active research, as the pathological roles involved are complex and not yet fully understood, and the related therapeutic approaches are not well developed. In this review, we describe insulin resistance and add recent advances in the major pathological and physiological changes and underlying mechanisms by which insulin resistance leads to myocardial remodeling and dysfunction in the diabetic heart, including exosomal dysfunction, ferroptosis, and epigenetic factors. In addition, we discuss potential therapeutic approaches to improve insulin resistance and accelerate the development of cardiovascular protection drugs.

Breast cancer arises from a series of molecular alterations that disrupt cell cycle checkpoints, leading to aberrant cell proliferation and genomic instability. Targeted pharmacological inhibition of cell cycle regulators has long been considered a promising anti‐cancer strategy. Initial attempts to drug critical cell cycle drivers were hampered by poor selectivity, modest efficacy and haematological toxicity. Advances in our understanding of the molecular basis of cell cycle disruption and the mechanisms of resistance to CDK4/6 inhibitors have reignited interest in blocking specific components of the cell cycle machinery, such as CDK2, CDK4, CDK7, PLK4, WEE1, PKMYT1, AURKA and TTK. These targets play critical roles in regulating quiescence, DNA replication and chromosome segregation. Extensive preclinical data support their potential to overcome CDK4/6 inhibitor resistance, induce synthetic lethality or sensitise tumours to immune checkpoint inhibitors. This review provides a biological and drug development perspective on emerging cell cycle targets and novel inhibitors, many of which exhibit favourable safety profiles and promising activity in clinical trials. [ABSTRACT FROM AUTHOR]

Cancer is caused by a complex interaction of factors that interrupt the normal growth and division of cells. At the center of this process is the intricate relationship between DNA damage and the cellular mechanisms responsible for maintaining genomic stability. When DNA damage is not repaired, it can cause genetic mutations that contribute to the initiation and progression of cancer. On the other hand, the DNA damage response system, which involves the phosphorylation of the histone variant H2AX (γH2AX), is crucial in preserving genomic integrity by signaling and facilitating the repair of DNA double-strand breaks. This review provides an explanation of the molecular dynamics of H2AX in the context of DNA damage response. It emphasizes the crucial role of H2AX in recruiting and localizing repair machinery at sites of chromatin damage. The review explains how H2AX phosphorylation, facilitated by the master kinases ATM and ATR, acts as a signal for DNA damage, triggering downstream pathways that govern cell cycle checkpoints, apoptosis, and the cellular fate decision between repair and cell death. The phosphorylation of H2AX is a critical regulatory point, ensuring cell survival by promoting repair or steering cells towards apoptosis in cases of catastrophic genomic damage. Moreover, we explore the therapeutic potential of targeting H2AX in cancer treatment, leveraging its dual function as a biomarker of DNA integrity and a therapeutic target. By delineating the pathways that lead to H2AX phosphorylation and its roles in apoptosis and cell cycle control, we highlight the significance of H2AX as both a prognostic tool and a focal point for therapeutic intervention, offering insights into its utility in enhancing the efficacy of cancer treatments.

Abstract Breast cancer arises from a series of molecular alterations that disrupt cell cycle checkpoints, leading to aberrant cell proliferation and genomic instability. Targeted pharmacological inhibition of cell cycle regulators has long been considered a promising anti‐cancer strategy. Initial attempts to drug critical cell cycle drivers were hampered by poor selectivity, modest efficacy and haematological toxicity. Advances in our understanding of the molecular basis of cell cycle disruption and the mechanisms of resistance to CDK4/6 inhibitors have reignited interest in blocking specific components of the cell cycle machinery, such as CDK2, CDK4, CDK7, PLK4, WEE1, PKMYT1, AURKA and TTK. These targets play critical roles in regulating quiescence, DNA replication and chromosome segregation. Extensive preclinical data support their potential to overcome CDK4/6 inhibitor resistance, induce synthetic lethality or sensitise tumours to immune checkpoint inhibitors. This review provides a biological and drug development perspective on emerging cell cycle targets and novel inhibitors, many of which exhibit favourable safety profiles and promising activity in clinical trials.

Background: Outcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of TP53-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with TP53 mutations, has not been definitively evaluated. Methods: Anti-leukemic effects of adavosertib were investigated using a relapsed TP53 isogenic cell model system, primary patient, and patient-derived ALL samples (n = 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively. Results: Our study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (n = 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of TP53 mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL. Conclusions: Together, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL. [ABSTRACT FROM AUTHOR]

Purpose: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation. Conclusions: Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance antitumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment. [ABSTRACT FROM AUTHOR]

The expression of human papillomavirus (HPV) oncoproteins perturbed multiple cellular events of the host cells, leading to the formation of cancer phenotypes. Our current and previous studies indicated that Aurora kinase A (AurA), a mitotic regulator that is often aberrantly expressed in human cancers, is preferentially bound to E6-encoded by cancer-causing HPV. AurA is believed to be important for the proliferation and survival of HPV-positive cells. Nonetheless, the interaction between AurA and E6, and the mechanism of how this association is involved in carcinogenesis, have not been elucidated clearly. Hence, we performed a series of biochemical assays to characterize the AurA-E6 association and complex formation. We found the C-terminus of E6, upstream of the PDZ binding motif of E6, is important to forming the AurA-E6 complex in the nucleus. We also showed that the expression level of E6 corresponded positively with AurA expression. Meanwhile, the functional consequences of the AurA-E6 association to AurA kinase function and host cellular events were also delineated. Intriguingly, we revealed that AurA-E6 association regulated the expression of cyclin E and phosphor-Histone H3, which are involved in G1/S and mitotic phases of the cell cycle, respectively. Depletion of AurA also reduced the invasive ability of HPV-positive cells. AurA inhibition may not be sufficient to reduce the oncogenic potential exerted by E6. Altogether, our study unleashed the mechanism of how HPVE6 deploy AurA to promote cancer phenotypes, particularly through dysregulation of cell cycle checkpoints and suggests that the AurA-E6 complex possesses a therapeutic value. IMPORTANCE We unveiled the mechanism of how HPV employs Aurora kinase A (AurA) of host cells to exert its oncogenic capability synergistically. We systematically characterized the mode of interaction between E6-encoded by cancer-causing HPV and AurA. Then, we delineated the consequences of AurA-E6 complex formation on AurA kinase function and changes to cellular events at molecular levels. Using a cellbased approach, we unleashed that disruption of AurA-E6 association can halt cancer phenotype exhibited by HPV-positive cancer cells. Our findings are vital for the designing of state-of-the-art therapies for HPV-associated cancers. [ABSTRACT FROM AUTHOR]

Objective(s): Gemcitabine is a first-line drug for the treatment of bladder cancer. One of the most important mechanisms of gemcitabine resistance is the low expression of cellular membrane transporter hENT1. Various derivatives containing fatty acid side chains have been developed in order to facilitate gemcitabine uptake and prolong its retention in cells, such as CP-4126. In this study, the anti-tumor effect and mechanism of a new derivative of gemcitabine named SZY-200 on bladder cancer cells were investigated. SZY-200 was assembled from the gemcitabine-lauric acid conjugate.Materials and Methods: Antiproliferative activities of SZY-200 and lauric acid were evaluated using CCK-8 assay and clonogenic survival assay. The hENT1 inhibitor NBMPR was employed to determine the role of hENT1 in the apoptotic activity of GEM, CP-4126, and SZY-200. RT-qPCR, flow cytometry, fluorescence microscope, western blotting, and wound healing assay were used to study the mechanisms of SZY-200. The target genes were predicted using the BATMAN-TCM database.Results: Our data showed that SZY-200 could inhibit the proliferation of bladder cancer cells by inducing cell cycle arrest and apoptosis. The inhibitory effects were comparable to gemcitabine and CP-4126. SZY-200 does not rely on hENT1 to help it enter bladder cancer cells. Also, we found that lauric acid could inhibit the proliferation of bladder cancer cells. SZY-200 could down-regulate the expressions of PPARG and PTGS2 which were related to the occurrence and development of bladder cancer.Conclusion: SZY-200 has the same or more advantages as CP-4126 and could be an ideal candidate drug for further in vivo investigation.

Multiple myeloma (MM) is a blood cancer defined by the accumulation of abnormal plasma cells in the bone marrow. Bortezomib is still one of the most effectual anti-cancer drugs used for the treatment of MM. However, drug-resistance prevents the efficacy of chemotherapy. One of the mechanisms causing drug-resistance is changes in regulatory factors involved cell-cycle-checkpoints. In this study, the expression levels of cyclin-D1, cyclin-D2, cyclin-A1, cyclin-E1 and cyclin-B1 genes in bortezomib-sensitive (KMS-28)/resistant (KMS-20) multiple myeloma cell lines and their effects on resistance were investigated. MTT-assay was performed to determine bortezomib cytotoxicity in both cell lines. RNA isolation and cDNA synthesis were done from bortezomib treated cells. Expressions of the genes were analyzed by Real-Time-qPCR. The results of the study showed that cyclin-A1 was not expressed in bortezomib-sensitive cells, but overexpressed in bortezomib-resistant cells. This study was the first to demonstrate a direct role of cyclin-A1 at the development of bortezomib-resistance in MM. It was observed that cyclin-D1 expression increased in KMS-28 and decreased in KMS-20 cells. The results of the study suggest that suppression of cyclin-D1 in KMS-20 cells prevents apoptosis formation and therefore causes resistance. However, we found no evidence of an association of the cyclin-D2, cyclin-E1 and cyclin-B1 genes with the development of resistance. Consequently, the findings of the study will enable us to better understand MM disease at the molecular level and to develop new treatment strategies to prevent bortezomib resistance. [ABSTRACT FROM AUTHOR]

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~ 800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for finetuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC) arises in peribronchial locations and infiltrates the bronchial submucosa, including about 15% of lung cancer cases. Despite decades of research, the prognosis for SCLC patients remains poor because this tumor is characterized by an exceptionally high proliferative rate, strong tendency for early widespread metastasis and acquired chemoresistance. Omics profiling revealed that SCLC harbor extensive chromosomal rearrangements and a very high mutation burden. This led to the development of immune-checkpoint inhibitors as single agents or in combination with chemotherapy, which however resulted in a prolonged benefit only for a small subset of patients. Thus, the present review discusses the rationale and limitations of immunotherapeutic approaches, presenting the current biological understanding of aberrant signaling pathways that might be exploited with new potential treatments. In particular, new agents targeting DNA damage repair, cell cycle checkpoint, and apoptosis pathways showed several promising results in different preclinical models. Epigenetic alterations, gene amplifications and mutations can act as biomarkers in this context. Future research and improved clinical outcome for SCLC patients will depend on the integration between these omics and pharmacological studies with clinical translational research, in order to identify specific predictive biomarkers that will be hopefully validated using clinical trials with biomarker-selected targeted treatments. [ABSTRACT FROM AUTHOR]

Various stem cells in the body are tasked with maintaining tissue homeostasis throughout the life of an organism and thus must be resilient to intrinsic and extrinsic challenges such as infection and injury. Crucial to these challenges is genome maintenance because a high mutational load and persistent DNA lesions impact the production of essential gene products at proper levels and compromise optimal stem cell renewal and differentiation. Genome maintenance requires a robust and well-regulated DNA damage response suited to maintaining specific niches and tissues. In this review, we explore the similarities and differences between diverse stem cell types derived from (or preceding) all germ layers, including extraembryonic tissues. These cells utilize different strategies, including implementation of robust repair mechanisms, modulation of cell cycle checkpoints best suited to eliminating compromised cells, minimization of cell divisions, and differentiation in response to excessive damage. [ABSTRACT FROM AUTHOR]

Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiationinduced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling. [ABSTRACT FROM AUTHOR]

DNA replication ensures the complete and accurate duplication of the genome. The traditional approach to analysing perturbation of DNA replication is to use chemical inhibitors, such as hydroxyurea or aphidicolin, that slow or stall replication fork progression throughout the genome. An alternative approach is to perturb replication at a single site in the genome that permits a more forensic investigation of the cellular response to the stalling or disruption of a replication fork. This has been achieved in several organisms using different systems that share the common feature of utilizing the high affinity binding of a protein to a defined DNA sequence that is integrated into a specific locus in the host genome. Protein-mediated replication fork blocking systems of this sort have proven very valuable in defining how cells cope with encountering a barrier to fork progression. In this review, we compare protein-based replication fork barrier systems from different organisms that have been developed to generate site-specific replication fork perturbation. • Cells use DNA replication fork barriers to coordinate replication and transcription • Replication fork barriers created by tightly-bound proteins can be programmed or inadevertent. • Replication fork associated proteins can recognize some protein barriers and arrest replication • Many replication fork barriers have been moved to ectopic sites or to another organism to analyze their effect [ABSTRACT FROM AUTHOR]

Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses. G2 checkpoint abrogation after co-treatment with radiation and ATR inhibitors was accompanied by increased radiation-induced IFN signaling in four out of five cell lines tested. Consistent with the hypothesis that the cytosolic DNA sensor cGAS may detect DNA from ruptured micronuclei after G2 checkpoint abrogation, cGAS co-localized with micronuclei, and depletion of cGAS or STING abolished the IFN responses. Contrastingly, one lung cancer cell line showed no increase in IFN signaling despite irradiation and G2 checkpoint abrogation. This cell line showed a higher level of the exonuclease TREX1 than the other cell lines, but TREX1 depletion did not enhance IFN signaling. Rather, addition of a pan-caspase inhibitor restored the IFN response in this cell line and also increased the responses in the other cell lines. These results show that treatment-induced caspase activation can suppress the IFN response after co-treatment with radiation and ATR inhibitors. Caspase activation thus warrants further consideration as a possible predictive marker for lack of IFN signaling.

Background: The aberrant regulation of cell cycle is significantly correlated with cancer carcinogenesis and progression, in which cell cycle checkpoints control phase transitions, cell cycle entry, progression, and exit. However, the integrative role of cell cycle checkpoint-related genes (CRGs) in bladder carcinoma (BC) remains unknown. Methods: The transcriptomic data and clinical features of BC patients were downloaded from The Cancer Genome Atlas (TCGA), used to identify CRGs correlated with overall survival (OS) by univariate Cox regression analysis. Then, the multivariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses further developed a prognostic CRG signature, which was validated in three external datasets retrieved from Gene Expression Omnibus (GEO). The receiver operating characteristic curve (ROC) analysis was conducted for evaluating the performance of the CRG signature in prognosis prediction. RNA sequencing (RNA-Seq) was performed to explore the expression difference in the identified CRGs between tumor and normal tissue samples from 11 BC patients in the local cohort. Ultimately, genomic profiles and tumor microenvironment (TME), and the Genomics of Drug Sensitivity in Cancer (GDSC) were investigated to guide precision treatment for BC patients with different CRG features. Results: The novel constructed 23-CRG prognostic signature could stratify BC patients into high-risk and low-risk groups with significantly different outcomes (median OS: 13.64 vs. 104.65 months). Notably, 19 CRGs were the first to be identified as being associated with BC progression. In three additional validation datasets (GSE13507, GSE31684, and GSE32548), higher CRG scores all indicated inferior survival, demonstrating the robust ability of the CRG signature in prognosis prediction. Moreover, the CRG signature as an independent prognostic factor had a robust and stable risk stratification for BC patients with different histological or clinical features. Then, a CRG signature-based nomogram with a better performance in prognostic prediction [concordance index (C-index): 0.76] was established. Functional enrichment analysis revealed that collagen-containing extracellular matrix (ECM), and ECM-related and MAPK signaling pathways were significantly associated with the signature. Further analysis showed that low-risk patients were characterized by particularly distinctive prevalence of FGFR3 (17.03% vs. 6.67%, p < 0.01) and POLE alterations (7.97% vs. 2.50%, p < 0.05), and enrichment of immune infiltrated cells (including CD8+ T cells, CD4+ naïve T cells, follicular helper T cells, Tregs, and myeloid dendritic cells). RNA-seq data in our local cohort supported the findings in the differentially expressed genes (DEGs) between tumor and normal tissue samples, and the difference in TME between high-risk and low-risk groups. Additionally, CRG signature score plus FGFR3 status divided BC patients into four molecular subtypes, with distinct prognosis, TME, and transcriptomic profiling of immune checkpoint genes. Of note, CRG signature score plus FGFR3 status could successfully distinguish BC patients who have a higher possibility of response to immunotherapy or chemotherapy drugs. Conclusions: The CRG signature is a potent prognostic model for BC patients, and in combination with FGFR3 alterations, it had more practical capacity in the prediction of chemotherapy and immunotherapy response, helping guide clinical decision-making. [ABSTRACT FROM AUTHOR]

ABSTRACT Cell division in Escherichia coli starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, only after a considerable lag period does the cell start to form a midcell constriction. The onset of constriction depends upon the arrival of so-called late divisome proteins, among which, FtsN is the last essential one. The timing and dependency of FtsN arrival to the divisome, along with genetic evidence, suggests it triggers cell division. In this study, we used high-throughput fluorescence microscopy to determine the arrival of FtsN and the early divisome protein ZapA to midcell at a single-cell level during the cell cycle. Our data show while the recruitment of ZapA/FtsZ is gradual in the cell cycle, recruitment of FtsN is rapid and begins at about the onset of constriction. At this time, the fraction of ZapA/FtsZ in the Z ring approaches its peak value. We also find a second increase in FtsN recruitment to the divisome, which begins once the amount of ZapA/FtsZ at midcell starts decreasing. Increasing hypermorphic FtsA* (FtsA R286W), but not FtsA, accelerates FtsN recruitment but not constriction. This finding is consistent with FtsA* recruiting FtsN with some other divisome component being rate-limiting for constriction under these conditions. Finally, our data support the recently proposed idea that ZapA/FtsZ and FtsN are part of physically separate complexes in midcell throughout the whole septation process. IMPORTANCE Cell division in most bacteria starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, cells only start to constrict after a considerable lag. A factor thought to trigger the onset of constriction in Escherichia coli is FtsN, which is the last essential protein to be recruited to the Z ring. Using a high-throughput quantitative fluorescence microscopy, we determine the cell cycle-dependent recruitment of FtsN to the Z ring. Our data show rapid accumulation of FtsN to the Z ring about a quarter of the cell cycle after the formation of the Z ring. This initial wave is followed by another increase in FtsN recruitment once the FtsZ protofilament network starts to disassemble. The presence of FtsA* accelerates FtsN recruitment to the Z ring but does not lead to earlier constrictions. Our data furthermore suggest FtsZ and FtsN are part of physically separate complexes throughout the division process.

Lung adenocarcinoma (LUAD) is one of the most common malignancies with the highest mortality globally, and it has a poor prognosis. Cell cycle checkpoints play a central role in the entire system of monitoring cell cycle processes, by regulating the signalling pathway of the cell cycle. Cell cycle checkpoints related genes (CCCRGs) have potential utility in predicting survival, and response to immunotherapies and chemotherapies. To examine this, based on CCCRGs, we identified two lung adenocarcinoma subtypes, called cluster1 and cluster2, by consensus clustering. Enrichment analysis revealed significant discrepancies between the two subtypes in gene sets associated with cell cycle activation and tumor progression. In addition, based on Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression, we have developed and validated a cell cycle checkpoints-related risk signature to predict prognosis, tumour immune microenvironment: (TIME), immunotherapy and chemotherapy responses for lung adenocarcinoma patients. Results from calibration plot, decision curve analysis (DCA), and time-dependent receiver operating characteristic curve (ROC) revealed that combining age, gender, pathological stages, and risk score in lung adenocarcinoma patients allowed for a more accurate and predictive nomogram. The area under curve for lung adenocarcinoma patients with 1-, 3-, 5-, and 10-year overall survival was: 0.74, 0.73, 0.75, and 0.81, respectively. Taken together, our proposed 4-CCCRG signature can serve as a clinically useful indicator to help predict patients outcomes, and could provide important guidance for immunotherapies and chemotherapies decision for lung adenocarcinoma patients. [ABSTRACT FROM AUTHOR]

Abstract Background The temporal coordination of biological processes by the circadian clock is an important mechanism, and its disruption has negative health outcomes, including cancer. Experimental and theoretical evidence suggests that the oscillators driving the circadian clock and the cell cycle are coupled through phase locking. Results We present a detailed and documented map of known mechanisms related to the regulation of the circadian clock, and its coupling with an existing cell cycle map which includes main interactions of the mammalian cell cycle. The coherence of the merged map has been validated with a qualitative dynamics analysis. We verified that the coupled circadian clock and cell cycle maps reproduce the observed sequence of phase markers. Moreover, we predicted mutations that contribute to regulating checkpoints of the two oscillators. Conclusions Our approach underlined the potential key role of the core clock protein NR1D1 in regulating cell cycle progression. We predicted that its activity influences negatively the progression of the cell cycle from phase G2 to M. This is consistent with the earlier experimental finding that pharmacological activation of NR1D1 inhibits tumour cell proliferation and shows that our approach can identify biologically relevant species in the context of large and complex networks.

Lung adenocarcinoma (LUAD) is one of the most common malignancies with the highest mortality globally, and it has a poor prognosis. Cell cycle checkpoints play a central role in the entire system of monitoring cell cycle processes, by regulating the signalling pathway of the cell cycle. Cell cycle checkpoints related genes (CCCRGs) have potential utility in predicting survival, and response to immunotherapies and chemotherapies. To examine this, based on CCCRGs, we identified two lung adenocarcinoma subtypes, called cluster1 and cluster2, by consensus clustering. Enrichment analysis revealed significant discrepancies between the two subtypes in gene sets associated with cell cycle activation and tumor progression. In addition, based on Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression, we have developed and validated a cell cycle checkpoints-related risk signature to predict prognosis, tumour immune microenvironment: (TIME), immunotherapy and chemotherapy responses for lung adenocarcinoma patients. Results from calibration plot, decision curve analysis (DCA), and time-dependent receiver operating characteristic curve (ROC) revealed that combining age, gender, pathological stages, and risk score in lung adenocarcinoma patients allowed for a more accurate and predictive nomogram. The area under curve for lung adenocarcinoma patients with 1-, 3-, 5-, and 10-year overall survival was: 0.74, 0.73, 0.75, and 0.81, respectively. Taken together, our proposed 4-CCCRG signature can serve as a clinically useful indicator to help predict patients outcomes, and could provide important guidance for immunotherapies and chemotherapies decision for lung adenocarcinoma patients.

Recent developments in clinical patient-based personalized genomics explored a variety of biomarkers for diagnosis, prognosis, and therapy in breast invasive carcinoma (BRIC). BRCA1 mutations mediated a catastrophic situation for a damage-repairing apparatus that induced malignant transformation of breast tissue. To identify an association between BRCA1 regulatory behavior and the pathway-level proteome for determining drug discovery channels, here we developed a computational scheme for BRCA1 pathway dataset retrieval from PathCards: PATHWAY UNIFICATION DATABASE (1,073 superpaths of 3,215 human pathways from 12 sources), BRCA1 pathway regulation analysis from cBioPortal for Cancer Genomics (more than 40 datasets of above 13,000 cancer samples), and BRC1 network construction from STRING v11.0 database (24,584,628 proteins of 5,090 organisms). Our study reveals about 700 alterations of 64 pathway components in 482 BRIC samples, in which there were 422 loss-of-function (LOF) mutations and 278 amplifications. We found 19 members (BRCA1, BRCA2, FANCA, ATM, NBN, SMARCD2, HDAC9, PLK1, SMARCA4, POU2F1, TP53, HDAC2, HLTF, BLM, E2F4, UBC, E2F5, MRE11, and RB1) based on a minimum 2% participation that showed 541 alterations in which there were 193 amplifications and 348 LOF mutations. From 19 components, BRCA1, BRCA2, FANCA, ATM, and TP53 have high-level LOF mutations whereas E2F5, NBN, SMARCD2, and POU2F1 have prominent amplifications. We developed three modules in which the BRCA1 module has 12 members that are involved in damage sensing and repairing processes as hot spots showing overexpression and LOF mutations. This in silico approach uniquely addressed BRCA1 mutations’ influence on BRIC at the pathway proteome level, with module identification as a hub for drug designing. We proposed these proteins as biomarkers for first-class diagnosis and clinical investigations. In the future, BRCA1 pathway-related therapeutic markers are used for further experimental investigations regarding drug development in breast cancer biology.

Cancer is caused by a complex interaction of factors that interrupt the normal growth and division of cells. At the center of this process is the intricate relationship between DNA damage and the cellular mechanisms responsible for maintaining genomic stability. When DNA damage is not repaired, it can cause genetic mutations that contribute to the initiation and progression of cancer. On the other hand, the DNA damage response system, which involves the phosphorylation of the histone variant H2AX (γH2AX), is crucial in preserving genomic integrity by signaling and facilitating the repair of DNA double-strand breaks. This review provides an explanation of the molecular dynamics of H2AX in the context of DNA damage response. It emphasizes the crucial role of H2AX in recruiting and localizing repair machinery at sites of chromatin damage. The review explains how H2AX phosphorylation, facilitated by the master kinases ATM and ATR, acts as a signal for DNA damage, triggering downstream pathways that govern cell cycle checkpoints, apoptosis, and the cellular fate decision between repair and cell death. The phosphorylation of H2AX is a critical regulatory point, ensuring cell survival by promoting repair or steering cells towards apoptosis in cases of catastrophic genomic damage. Moreover, we explore the therapeutic potential of targeting H2AX in cancer treatment, leveraging its dual function as a biomarker of DNA integrity and a therapeutic target. By delineating the pathways that lead to H2AX phosphorylation and its roles in apoptosis and cell cycle control, we highlight the significance of H2AX as both a prognostic tool and a focal point for therapeutic intervention, offering insights into its utility in enhancing the efficacy of cancer treatments. • The histone variation H2AX is essential to the DNA damage response and offers a potential cancer therapeutic target. • H2AX is a well-known chromatin modification event in DNA damage response and human malignancies. • H2AX phosphorylation (γ-H2AX) is necessary for DNA repair protein assembly at damaged chromatin regions. • Detecting double strand breaks with phosphorylated histone H2AX may help in cancer treatment. [ABSTRACT FROM AUTHOR]

Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP‐dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate‐limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single‐stranded DNA, providing the signal for the activation of the Mec1/ATR–Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes. Synopsis: While nucleotide metabolism is known to be tightly linked to DNA replication and repair, this work addresses more general yeast metabolome remodeling in response to replication stress, revealing a role for SNF1/AMPK in maintenance of a reductive cellular milieu that facilitates ssDNA binding by RPA. Hydroxyurea treatment diverts glucose into the oxidative pentose phosphate pathway.Sugar metabolism rewiring in response to replication stress is mediated by AMP‐dependent kinase SNF1.SNF1 phosphorylates transcription factor Mig1 to derepress ZWF1/G6PD transcription.Cellular redox state regulates RPA loading and S‐phase checkpoint activation. [ABSTRACT FROM AUTHOR]

Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress. A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated. : 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [ https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2%5ftargetmap%5fv1 ]. • Systematic literature research of replication stress as a target for treatment. • Focus on the six specific drug targets ATM, ATR, CHK1, DNA-PK, PARP and WEE1. • ATR, CHK1, PARP and WEE1 are the most promising targets using mono/combination therapy. • Dual targeting of replication stress pathways shows potential in novel combinations. [ABSTRACT FROM AUTHOR]

ABSTRACT Cytomegaloviruses (CMVs) are among the largest pathogenic viruses in mammals. To enable replication of their long double-stranded DNA genomes, CMVs induce profound changes in cell cycle regulation. A hallmark of CMV cell cycle control is the establishment of an unusual cell cycle arrest at the G1/S transition, which is characterized by the coexistence of cell cycle stimulatory and inhibitory activities. While CMVs interfere with cellular DNA synthesis and cell division, they activate S-phase-specific gene expression and nucleotide metabolism. This is facilitated by a set of CMV gene products that target master regulators of G1/S progression such as cyclin E and A kinases, Rb-E2F transcription factors, p53-p21 checkpoint proteins, the APC/C ubiquitin ligase, and the nucleotide hydrolase SAMHD1. While the major themes of cell cycle regulation are well conserved between human and murine CMVs (HCMV and MCMV), there are considerable differences at the level of viral cell cycle effectors and their mechanisms of action. Furthermore, both viruses have evolved unique mechanisms to sense the host cell cycle state and modulate the infection program accordingly. This review provides an overview of conserved and divergent features of G1/S control by MCMV and HCMV.

Background: Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable due to old age or advanced stage; thus, radio- and chemotherapy are considered the standard treatments for these patients. However, due to the radiation resistance of tumor cells that may arise during radiotherapy, results are still not satisfactory. The authors' previous studies found that microRNA can affect radiosensitivity, and further microRNA research was conducted to improve the radiosensitivity of ESCC.Methods: Cells were treated with silent miR-29b (si-miR-29b). Thereafter,proliferation, colony formation, cell cycle, and apoptosis were determined. The luciferase reporting assay was used to confirm the direct interaction between miR-29b and BTG2. Serum samples and clinical follow-up data of 75 elderly or advanced ESCC patients who could not tolerate surgery were collected.Results: The expression level of miR-29 in ESCC serum was closely correlated to radiosensitivity (χ2 =8.36, p < 0.05) and correlated with overall survival (OS; hazard ratio [HR] 0.47, 95% confidence interval [CI] 0.24-0.90). Function assays demonstrated that the number of cell clones increased after radiometry radiation, and the cell cycle was blocked in the G0/G1 phase (from 37.2 to 56.9%) in the si-miR-29b transfection group. Expression of BTG2 was upregulated and expression of cyclin D1 was downregulated (p < 0.05). Transfection of si-BTG2 can reverse this result and restore the expression level of cyclin D1 (p < 0.05). The target gene BTG2 of miR-29b was predicted using a bioinformatics tool and confirmed by dual-luciferase reporter assay.Conclusion: Silencing of miR-29b in ESCC cells can increase expression of BTG2 and decrease the level of intracellular cyclin D1, resulting in cell cycle arrest and accumulation in the G0/G1 phase. Because G0/G1-phase cells are insensitive to radiotherapy, the sensitivity of radiotherapy is reduced. [ABSTRACT FROM AUTHOR]

Abstract: FITOPROT, which contains curcuminoids and Bidens pilosa L. extract, is an innovative mucoadhesive formulation indicated for the topical treatment of chemoradiotherapy-induced oral mucositis (OM) in patients with advanced and visible oral squamous cell carcinoma. The formulation is used as a mouthwash directly on tumor tissue of patients with advanced neoplasms, without triggering cancer cell proliferation or tumor invasiveness. Thus, the aim of this study was to evaluate the biological effects of FITOPROT on an oral squamous cell carcinoma cell line (SCC-4). The viability of SCC-4 cells was assessed after exposure to FITOPROT using MTT reduction assay. The effects of the mucoadhesive formulation on cell cycle progression and cell death parameters were evaluated using flow cytometry. In addition, the inflammatory profile of the tumor cells was evaluated using the cytometric bead array (CBA) assay. FITOPROT promoted a concentration-dependent decrease in cell viability and cell cycle arrest at the G2/M phase (p < 0.05). Mitochondrial membrane potential was also altered after exposure to the formulation (p < 0.05), in parallel with a reduction in VEGF and IL-8 production (p = 0.01 and p = 0.05, respectively). In summary, the results indicate that FITOPROT reduces SCC-4 cell viability, promotes cell cycle arrest, modulates mitochondrial membrane potential, and exhibits antiangiogenic and anti-inflammatory properties, thus indicating its potential for topical use in patients with OM and visible tumors in the mouth.

Background: The temporal coordination of biological processes by the circadian clock is an important mechanism, and its disruption has negative health outcomes, including cancer. Experimental and theoretical evidence suggests that the oscillators driving the circadian clock and the cell cycle are coupled through phase locking. Results: We present a detailed and documented map of known mechanisms related to the regulation of the circadian clock, and its coupling with an existing cell cycle map which includes main interactions of the mammalian cell cycle. The coherence of the merged map has been validated with a qualitative dynamics analysis. We verified that the coupled circadian clock and cell cycle maps reproduce the observed sequence of phase markers. Moreover, we predicted mutations that contribute to regulating checkpoints of the two oscillators. Conclusions: Our approach underlined the potential key role of the core clock protein NR1D1 in regulating cell cycle progression. We predicted that its activity influences negatively the progression of the cell cycle from phase G2 to M. This is consistent with the earlier experimental finding that pharmacological activation of NR1D1 inhibits tumour cell proliferation and shows that our approach can identify biologically relevant species in the context of large and complex networks. [ABSTRACT FROM AUTHOR]

• A synthetic lethality between DNA damage and DNA damage defense is reported. • Hybrid prodrug nanoassembly is developed for dual drug co-delivery. • The reported synthetic lethality induces tumor apoptosis by mitotic catastrophe. Although DNA damage-based chemotherapy has been well investigated for clinical oncology, their effectiveness is greatly hindered by the DNA damage defenses including DNA damage repair and cell cycle arrest. Herein, we propose hybrid prodrug nanoassemblies using core-matched strategy and self-assembly technology to facilitate a synthetic lethal relationship between DNA damage and DNA damage defense. Hydroxycamptothecin (HCPT), a topoisomerase I inhibitor, is chosen as the DNA toxic chemotherapeutic. Norcantharidin (NCTD), a PP2A inhibitor, serves to abrogate homologous recombination repair and G 2 /M cell cycle arrest initiated by HCPT. Hybrid prodrug nanoassemblies efficiently co-deliver the two drugs with notably different physicochemical properties and lay solid foundation for their synthetic lethal synergy. The developed synthetic lethal synergy potently damages DNA and successfully attenuates DNA damage defense. More intriguingly, such synthetic lethality synergy induces tumor apoptosis by the cell death mechanism of mitotic catastrophe. In both breast tumor and colon tumor mouse models, hybrid prodrug nanoassemblies remarkably suppresses tumor proliferation and improves the safety of chemotherapy. Our results provide a promising strategy to potentiate DNA damage-based translational medicine. [ABSTRACT FROM AUTHOR]

Abstract Background Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance. Methods We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks. Results Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance. Conclusions Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.

Human kinome/phosphatome screen identified CAMK2N1 genes suppressing the development of human hepatocellular carcinoma (HCC). CAMK2N1 downregulation was found in 47% HCCs and associated with poor prognosis. The downregulation was mainly attributed to its genome deletion (28.4%) and DNA hypermethylation of its promoter (12.5%). Silencing and ectopic expression of CAMK2N1 respectively enhanced and suppressed cell proliferation, colony formation, and xenograft tumor growth in nude mice. Comparative proteomics revealed that CAMK2N1 silencing transcriptionally deregulated the genes regulated by E2F1 (89 out of the 114 E2F-signaling targets, P = 8.8E-240). The promoter assays revealed that CAMK2N1 suppressed E2F1-mediated transcriptional activities. CAMK2N1 silencing induced cyclins D/E expression, whereas its ectopic expression induced P27/KIP1 expression and suppressed the cell cycle. CAMK2N1 was translocated from the nuclei to the cytoplasm when cell proliferation reached the stationary phase, where its functions as an endogenous inhibitor of CAMK2. In conclusion, CAMK2NA is a novel 1p36 tumor suppressor gene that inhibits E2F1 transcriptional activities and induces P27/KIP1 expression. CAMK2N1-CAMK2 signaling forms a mechanism that restricts the cell cycle progression. Its deregulation could lead to tumorigenesis and might serve as promising therapeutic targets. [ABSTRACT FROM AUTHOR]

FITOPROT, which contains curcuminoids and Bidens pilosa L. extract, is an innovative mucoadhesive formulation indicated for the topical treatment of chemoradiotherapy-induced oral mucositis (OM) in patients with advanced and visible oral squamous cell carcinoma. The formulation is used as a mouthwash directly on tumor tissue of patients with advanced neoplasms, without triggering cancer cell proliferation or tumor invasiveness. Thus, the aim of this study was to evaluate the biological effects of FITOPROT on an oral squamous cell carcinoma cell line (SCC-4). The viability of SCC-4 cells was assessed after exposure to FITOPROT using MTT reduction assay. The effects of the mucoadhesive formulation on cell cycle progression and cell death parameters were evaluated using flow cytometry. In addition, the inflammatory profile of the tumor cells was evaluated using the cytometric bead array (CBA) assay. FITOPROT promoted a concentration-dependent decrease in cell viability and cell cycle arrest at the G2/M phase (p < 0.05). Mitochondrial membrane potential was also altered after exposure to the formulation (p < 0.05), in parallel with a reduction in VEGF and IL-8 production (p = 0.01 and p = 0.05, respectively). In summary, the results indicate that FITOPROT reduces SCC-4 cell viability, promotes cell cycle arrest, modulates mitochondrial membrane potential, and exhibits antiangiogenic and anti-inflammatory properties, thus indicating its potential for topical use in patients with OM and visible tumors in the mouth. [ABSTRACT FROM AUTHOR]

Radiation therapy is an important part of the standard of care treatment of brain tumors. However, the efficacy of radiation therapy is limited by the radioresistance of tumor cells, a phenomenon held responsible for the dismal prognosis of the most aggressive brain tumor types. A promising approach to radiosensitization of tumors is the inhibition of cell cycle checkpoint control responsible for cell cycle progression and the maintenance of genomic integrity. Inhibition of the kinases involved in these control mechanisms can abolish cell cycle checkpoints and DNA damage repair and thus increase the sensitivity of tumor cells to radiation and chemotherapy. Here, we discuss preclinical progress in molecular targeting of ATM, ATR, CHK1, CHK2, and WEE1, checkpoint kinases in the treatment of brain tumors, and review current clinical phase I-II trials.

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Many biological features are conserved and thus considered to be resistant to evolutionary change. While rapid genetic adaptation following the removal of conserved genes has been observed, we often lack a mechanistic understanding of how adaptation happens. We used the budding yeast, Saccharomyces cerevisiae, to investigate the evolutionary plasticity of chromosome metabolism, a network of evolutionary conserved modules. We experimentally evolved cells constitutively experiencing DNA replication stress caused by the absence of Ctf4, a protein that coordinates the enzymatic activities at replication forks. Parallel populations adapted to replication stress, over 1000 generations, by acquiring multiple, concerted mutations. These mutations altered conserved features of two chromosome metabolism modules, DNA replication and sister chromatid cohesion, and inactivated a third, the DNA damage checkpoint. The selected mutations define a functionally reproducible evolutionary trajectory. We suggest that the evolutionary plasticity of chromosome metabolism has implications for genome evolution in natural populations and cancer.

Cancer cells adopt glycolysis to facilitate the generation of biosynthetic substrates demanded by cell proliferation and growth, and to adapt to stress conditions such as excessive reactive oxygen species (ROS) accumulation. TIGAR (TP53-induced glycolysis and apoptosis regulator) is a fructose-2,6-bisphosphatase that is regulated by p53. TIGAR functions to inhibit glycolysis and promote antioxidative activities, which assists the generation of NADPH to maintain the levels of GSH and thus reduces intracellular ROS. However, the functions of TIGAR in gastric cancer (GC) remain unclear. TIGAR expression levels were detected by immunoblotting and immunohistochemistry in gastric cancer samples, along with four established cell lines of GC. The functions of TIGAR were determined by utilizing shRNA-mediated knockdown experiments. The NADPH/NADP+ ratio, ROS, mitochondrial ATP production, and phosphorus oxygen ratios were determined in TIGAR-depleted cells. Xenograft experiment was conducted with BALB/c nude mice. TIGAR was up-regulated compared with corresponding non-cancerous tissues in primary GCs. TIGAR knockdown significantly reduced cell proliferation and increased apoptosis. TIGAR protected cancer cells from oxidative stress-caused damages, but also glycolysis defects. TIGAR also increased the production of NADPH in gastric cancer cells. TIGAR knockdown led to increased ROS production, elevated mitochondrial ATP production, and phosphorus oxygen ratios. The prognosis of high TIGAR expression patients was significantly poorer than those with low TIGAR expression. Taken together, TIGAR exhibits oncogenic features in GC, which can be evaluated as a target for intervention in the treatment of GC.

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Copyright of HNO is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of HNO is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cancer cells adopt glycolysis to facilitate the generation of biosynthetic substrates demanded by cell proliferation and growth, and to adapt to stress conditions such as excessive reactive oxygen species (ROS) accumulation. TIGAR (TP53-induced glycolysis and apoptosis regulator) is a fructose-2,6-bisphosphatase that is regulated by p53. TIGAR functions to inhibit glycolysis and promote antioxidative activities, which assists the generation of NADPH to maintain the levels of GSH and thus reduces intracellular ROS. However, the functions of TIGAR in gastric cancer (GC) remain unclear. TIGAR expression levels were detected by immunoblotting and immunohistochemistry in gastric cancer samples, along with four established cell lines of GC. The functions of TIGAR were determined by utilizing shRNA-mediated knockdown experiments. The NADPH/NADP+ ratio, ROS, mitochondrial ATP production, and phosphorus oxygen ratios were determined in TIGAR-depleted cells. Xenograft experiment was conducted with BALB/c nude mice. TIGAR was up-regulated compared with corresponding non-cancerous tissues in primary GCs. TIGAR knockdown significantly reduced cell proliferation and increased apoptosis. TIGAR protected cancer cells from oxidative stress-caused damages, but also glycolysis defects. TIGAR also increased the production of NADPH in gastric cancer cells. TIGAR knockdown led to increased ROS production, elevated mitochondrial ATP production, and phosphorus oxygen ratios. The prognosis of high TIGAR expression patients was significantly poorer than those with low TIGAR expression. Taken together, TIGAR exhibits oncogenic features in GC, which can be evaluated as a target for intervention in the treatment of GC. [ABSTRACT FROM AUTHOR]