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Objective: The aim of this research is to understand the role of microRNA in cell cycle regulation especially on G2M Checkpoint from Luminal A samples Indonesian population. The profile results are used as biomarkers and therapeutic targets for breast cancer. For this reason, analysis was carried out on the comparison of miRNA expression between Luminal A and Fibroadenoma  mamae (FAM) using Nanostring nCounter., Methods: In this study, 5 (Formalin-Fixed Paraffin-Embedded) FFPE Luminal A tissues and 4 FFPE FAM samples were used. RNA was isolated from cancer tissue samples. Differential expression analysis of miRNA was conducted using Nanostring nCounter technology, subsequently followed by the expression analysis between FAM and Luminal A using nSolver softwere. Elevated expression levels of miRNAs were subjected to pathway and gene regulation analysis using KEGG and GSEA MsigDB databases. Data visualization was performed utilizing Cytoscape, NetworkAnalyst, and SRplot tools., Result: Based on 792 miRNAs detected on Nanostring nCounter, it was found that 60 miRNAs were upregulated and 6 miRNAs were downregulated. The 15 upregulated miRNAs analyzed show their role in the G2M Checkpoint through several pathways. The five miRNAs that significantly regulate the G2M Checkpoint are hsa-miR-196b-5p, hsa-miR-218-5p, hsa-miR-7-5p, hsa-miR-19a-5p, and hsa-miR-18a-5p Where each of these miRNAs regulates the CDKN1B gene., Conclusion: Significant differences in the expression of multiple miRNAs between Luminal A and FAM samples were observed. Furthermore, several of these miRNAs were found to modulate the G2M Checkpoint in Luminal A cancer by suppressing tumor suppressor genes.

A-MYB (MYBL1) is a transcription factor with a role in meiosis in spermatocytes. The related B-MYB protein is a key oncogene and a master regulator activating late cell cycle genes. To activate genes, B-MYB forms a complex with MuvB and is recruited indirectly to cell cycle genes homology region (CHR) promoter sites of target genes. Activation through the B-MYB-MuvB (MMB) complex is essential for successful mitosis. Here, we discover that A-MYB has a function in transcriptional regulation of the mitotic cell cycle and can substitute for B-MYB. Knockdown experiments in cells not related to spermatogenesis show that B-MYB loss alone merely delays cell cycle progression. Only dual knockdown of B-MYB and A-MYB causes G2/M cell cycle arrest, endoreduplication, and apoptosis. A-MYB can substitute for B-MYB in binding to MuvB. The resulting A-MYB-MuvB complex activates genes through CHR sites. We find that A-MYB activates the same target genes as B-MYB. Many of the corresponding proteins are central regulators of the cell division cycle. In summary, we demonstrate that A-MYB is an activator of the mitotic cell cycle by activating late cell cycle genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.

UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest., (© 2023 Qi et al.)

The average survival of patients with glioblastoma is 12-15 months. Therefore, finding a new treatment method is important, especially in cases that show resistance to treatment. Extremely low-frequency electromagnetic fields (ELF-EMF) have characteristics and capabilities that can be proposed as a new cancer treatment method with low side effects. This research examines the antitumor effect of ELF-EMF on U87 and U251 glioblastoma cell lines. Flowcytometry determined the viability/apoptosis and distribution of cells in different phases of the cell cycle. The size of cells was assessed by TEM. Important cell cycle regulation genes mRNA expression levels were investigated by real-time PCR. ELF-EMF induced apoptosis in U87cells much more than U251 (15% against 2.43%) and increased G2/M cell population in U87 (2.56%, p value < 0.05), and S phase in U251 (2.4%) (data are normalized to their sham exposure). The size of U87 cells increased significantly after ELF-EMF exposure (overexpressing P53 in U251 cells increased the apoptosis induction by ELF-EMF). The expression level of P53, P21, and MDM2 increased and CCNB1 decreased in U87. Among the studied genes, MCM6 expression decreased in U251. Increasing expression of P53, P21 and decreasing CCNB1, induction of cell G2/M cycle arrest, and consequently increase in the cell size can be suggested as one of the main mechanisms of apoptosis induction by ELF-EMF; furthermore, our results demonstrate the possible footprint of P53 in the apoptosis induction by ELF-EMF, as U87 carry the wild type of P53 and U251 has the mutated form of this gene., (© 2023. The Author(s).)

Renal tubular epithelial cells (TECs) play a key role in kidney fibrosis by mediating cycle arrest at G2/M. However, the key HDAC isoforms and the underlying mechanism that are involved in G2/M arrest of TECs remain unclear. Here, we find that Hdac9 expression is significantly induced in the mouse fibrotic kidneys, especially in proximal tubules, induced by aristolochic acid nephropathy (AAN) or unilateral ureter obstruction (UUO). Tubule-specific deletion of HDAC9 or pharmacological inhibition by TMP195 attenuates epithelial cell cycle arrest in G2/M, then reduces production of profibrotic cytokine and alleviates tubulointerstitial fibrosis in male mice. In vitro, knockdown or inhibition of HDAC9 alleviates the loss of epithelial phenotype in TECs and attenuates fibroblasts activation through inhibiting epithelial cell cycle arrest in G2/M. Mechanistically, HDAC9 deacetylates STAT1 and promotes its reactivation, followed by inducing G2/M arrest of TECs, finally leading to tubulointerstitial fibrosis. Collectively, our studies indicate that HDAC9 may be an attractive therapeutic target for kidney fibrosis., (© 2023. The Author(s).)

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

DNA repair mechanisms keep genome integrity and limit tumor-associated alterations and heterogeneity, but on the other hand they promote tumor survival after radiation and genotoxic chemotherapies. We screened pathway activation levels of 38 DNA repair pathways in nine human cancer types (gliomas, breast, colorectal, lung, thyroid, cervical, kidney, gastric, and pancreatic cancers). We took RNAseq profiles of the experimental 51 normal and 408 tumor samples, and from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium databases - of 500/407 normal and 5752/646 tumor samples, and also 573 normal and 984 tumor proteomic profiles from Proteomic Data Commons portal. For all the samplings we observed a congruent trend that all cancer types showed inhibition of G2/M arrest checkpoint pathway compared to the normal samples, and relatively low activities of p53-mediated pathways. In contrast, other DNA repair pathways were upregulated in most of the cancer types. The G2/M checkpoint pathway was statistically significantly downregulated compared to the other DNA repair pathways, and this inhibition was strongly impacted by antagonistic regulation of (i) promitotic genes CCNB and CDK1, and (ii) GADD45 genes promoting G2/M arrest. At the DNA level, we found that ATM, TP53, and CDKN1A genes accumulated loss of function mutations, and cyclin B complex genes - transforming mutations. These findings suggest importance of activation for most of DNA repair pathways in cancer progression, with remarkable exceptions of G2/M checkpoint and p53-related pathways which are downregulated and neutrally activated, respectively., Competing Interests: Conflict of interest statement Authors Anton Buzdin and Andrew Garazha were employed by the company OmicsWay Corp., and Victor Tkachev, Andrew Garazha, and Maxim Sorokin were employed by the company Oncobox Ltd. The remaining authors have only academic or clinical affiliations. All the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Understanding the cellular mechanisms underlying chronic kidney disease (CKD) progression is required to develop effective therapeutic approaches. In this issue of the JCI, Taguchi, Elias, et al. explore the relationship between cyclin G1 (CG1), an atypical cyclin that induces G2/M proximal tubule cell cycle arrest, and epithelial dedifferentiation during fibrogenesis. While CG1-knockout mice were protected from fibrosis and had reduced G2/M arrest, protection was unexpectedly independent of induction of G2/M arrest. Rather, CG1 drove fibrosis by regulating maladaptive dedifferentiation in a CDK5-dependent mechanism. These findings highlight the importance of maladaptive epithelial dedifferentiation in kidney fibrogenesis and identify CG1/CDK5 signaling as a therapeutic target in CKD progression.

Background: The incidence of melanoma rapidly increased in the past decades, and the clinical treatment of melanoma met huge challenges because of tumor heterogeneity and drug resistance. C118P, a novel tubulin polymerization inhibitor, exhibited strong anticancer effects in many tumors. However, there was no data regarding the potential effects of C118P in melanoma cells., Objective: To investigate of the efficacy and potential target of C118P in melanoma cells., Methods: Human melanoma cells were treated with C118P, followed by assessments of proliferation, apoptosis and cell cycle distribution. Subsequently, RNA sequencing was performed to further identify the drug targets of C118P in melanoma cells. GO analysis and protein-protein interaction networks analysis were used to screen the potential targets, and verified by a series of assays. Finally, the anti-growth activity of C118P was evaluated in A375-xenografted nude mice, and the expression of BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B), Ki67 and Tunel were determined., Results: We found that C118P concentration-dependently inhibited proliferation of melanoma cells. Moreover, C118P simultaneously triggered dramatic G2/M arrest and apoptosis via independent mechanisms in melanoma cells in vitro. C118P exerted anti-melanoma effects by inducing potent G2/M arrest, which was mechanistically related to downregulation of the expression of BUB1B. Importantly, C118P inhibited the tumor growth in A375-xenografted nude, and increased the staining of Ki-67 and Tunel and suppressed the expression of BUB1B in melanoma tissues, which was consistent with in vitro study., Conclusion: C118P might provide a novel strategy for the clinical treatment of melanoma by inhibition of BUB1B., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

The mechanisms underlying fibrogenic responses after injury are not well understood. Epithelial cell cycle arrest in G2/M after injury is a key checkpoint for determining wound-healing leading to either normal cell proliferation or fibrosis. Here, we identify a kidney- and liver-enriched circular RNA, circBNC2, which is abundantly expressed in normal renal tubular cells and hepatocytes but significantly downregulated after acute ischemic or toxic insult. Loss of circBNC2 is at least partially mediated by upregulation of DHX9. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrate that circBNC2 acts as a negative regulator of cell G2/M arrest by encoding a protein that promotes formation of CDK1/cyclin B1 complexes. Restoring circBNC2 in experimentally-induced male mouse models of fibrotic kidney and liver, decreases G2/M arrested cell numbers with secretion of fibrotic factors, thereby mitigating extracellular matrix deposition and fibrosis. Decreased expression of circBNC2 and increased G2/M arrest of epithelial cells are recapitulated in human ischemic reperfusion injury (IRI)-induced chronic kidney disease and inflammation-induced liver fibrosis, highlighting the clinical relevance. These findings suggest that restoring circBNC2 might represent a potential strategy for therapeutic intervention in epithelial organ fibrosis., (© 2022. The Author(s).)

The transcription factor Wilms' tumor 1 (WT1) is involved in development, tissue homeostasis, and disease. However, the exact roles and the mechanisms of WT1 in renal carcinoma are not well understood. Therefore, in this study, we evaluated the ability of WT1 to block proliferation in renal carcinoma cells in vitro. Experimental analysis showed that WT1 overexpression inhibited the proliferation of renal carcinoma A498 cells and promoted arrest at the G2/M checkpoint. RNA-Seq identified differentially expressed genes, including IL-24, related to both the cell proliferation and the cell cycle. WT1 overexpression upregulated IL-24 expression, and IL-24 overexpression induced G2/M arrest. ChIP-Seq identified JUN as a direct target of WT1 in A498 cells, in which positive regulation was shown by RT-qPCR. It has been shown that the transcription factor JUN can regulate IL-24 expression, and therefore, we hypothesize that WT1 might regulate the IL-24 through JUN. Furthermore, analysis based on TCGA datasets showed that the expression of WT1-regulated genes, including TXNIP and GADD45A, was significantly correlated with the stage and histological grade of tumors, with high levels linked to favorable prognoses. Our results demonstrated that the overexpression of WT1 upregulates IL-24, leading to G2/M checkpoint arrest to reduce proliferation. These results indicate that regulation of IL-24 by WT1 inhibits proliferation and may represent a potential target for treating renal carcinoma., Competing Interests: The authors declare that there are no competing interests., (Copyright © 2022 Y. J. Jing et al.)

Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 μM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Acidic nucleoplasmic DNA binding protein 1 (AND-1, also known as WD repeat and HMG-box DNA-binding protein 1, WDHD1) plays an important role in DNA replication and repair, but the relationship between AND-1 and radiosensitivity is not well understood. This research explored the impact of AND-1 on the radiosensitivity of non-small cell lung cancer (NSCLC) for the first time. NSCLC cells were treated with AND-1 siRNA or a new AND-1 inhibitor, CH-3, and clonogenic survival assay was used to characterize cell radiosensitivity. Cell cycle and apoptosis were examined by flow cytometry. DNA damage was detected by comet assay, immunofluorescence, and homologous recombination (HR) repair assay. Finally, the radiosensitization effect of CH-3 was investigated in vivo in a xenograft tumor model. The results showed that AND-1 inhibition significantly increased the radiosensitivity of NSCLC cells. Mechanistically, AND-1 inhibitor (CH-3) induced G2/M phase arrest by regulating the ATM signaling pathway and enhanced irradiation-induced DNA damage by inhibiting the DNA HR repair pathway. CH-3 enhanced the radiosensitivity of NSCLC cells in vivo. The development of radiosensitizers that target AND-1 may provide an alternative strategy to inhibit NSCLC., (Copyright © 2022. Published by Elsevier B.V.)

We report a novel topoisomerase IIα inhibitor, mercaptopyridine oxide (MPO), which induces G2/M arrest and senescence with distinctly different cell cycle regulators (p21 or p14 ARF ) in HCT116p 53 WT and HCT116 p53 -/- cells, respectively. MPO treatment induced defective topoisomerase IIα-mediated decatenation process and inhibition of the enzyme's catalytic activity that stalled entry into mitosis. Topoisomerase IIα inhibition was associated with ROS-mediated activation of ATM-Chk2 kinase axis in HCT116 p53 WT cells, but not in HCT116 p53 -/- cells displaying early Chk1 activation. Results suggest that E2F1 stabilization might link MPO-induced p53 phospho-activation in HCT116 p53 WT cells or p14 ARF induction in HCT116 p53 -/- cells. Also, interaction between topoisomerase IIα and Chk1 was induced in both cell lines, which could be important for decatenation checkpoint activation, even upon p53 ablation. Notably, TCGA dataset analyses revealed topoisomerase IIα upregulation across a wide array of cancers, which was associated with lower overall survival. Corroborating that increased topoisomerase IIα expression might offer susceptibility to the novel inhibitor, MPO (5 μM) induced strong inhibition in colony forming ability of pancreatic and hepatocellular cancer cell lines. These data highlight a novel topoisomerase IIα inhibitor and provide proof-of-concept for its therapeutic potential against cancers even with loss-of-function of p53., (Copyright © 2021. Published by Elsevier B.V.)

Purpose: Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint., Methods and Materials: The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining., Results: RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR., Conclusions: RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients., (© 2022. The Author(s).)

Comprehensive genomic studies have delineated key driver mutations linked to disease progression for most cancers. However, corresponding transcriptional changes remain largely elusive because of the bias associated with cross-study analysis. Here, we overcome these hurdles and generate a comprehensive prostate cancer transcriptome atlas that describes the roadmap to tumor progression in a qualitative and quantitative manner. Most cancers follow a uniform trajectory characterized by upregulation of polycomb-repressive-complex-2, G2-M checkpoints, and M2 macrophage polarization. Using patient-derived xenograft models, we functionally validate our observations and add single-cell resolution. Thereby, we show that tumor progression occurs through transcriptional adaption rather than a selection of pre-existing cancer cell clusters. Moreover, we determine at the single-cell level how inhibition of EZH2 - the top upregulated gene along the trajectory - reverts tumor progression and macrophage polarization. Finally, a user-friendly web-resource is provided enabling the investigation of dynamic transcriptional perturbations linked to disease progression., (© 2021. The Author(s).)

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gliomas are the major type of primary brain tumors. Accumulating research has demonstrated that tubulin is connected with the development and malignant progression of tumors. TUBA1C is a subtype of α-tubulin and is linked to prognosis in multiple cancers. In this study, the prognosis-related gene TUBA1C in glioma was identified and analyzed by bioinformatic approaches such as Kaplan-Meier (KM) survival time analysis, univariate and multivariate Cox analysis, receiver operating characteristic (ROC) analysis and functional enrichment analysis. Based on the above analyses, we found that glioma tissues had significantly higher expression of TUBA1C than normal brain tissues, and high expression of TUBA1C has worse prognosis in glioma. Gene set enrichment analysis (GSEA) revealed the signaling pathways related to the cell cycle. Furthermore, knockdown of TUBA1C also inhibited proliferation and migration and caused apoptosis and G2/M phase arrest in glioma cells. This study demonstrated that high TUBA1C expression correlated with poor outcomes in glioma patients and that knocking down TUBA1C suppressed glioma cell proliferation via cell cycle arrest. In addition, TUBA1C might be a therapeutic biomarker for gliomas., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G 2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro . Using a copper-repressible promoter P CTR4 -CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G 2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G 2 arrest. Release from this G 2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G 2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ataxia telangiectasia mutated and rad3-related kinase (ATR)-dependent phosphorylation of checkpoint kinase 1 (Chk1) that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018; Kizhedathu et al., 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H 2 O 2 generating dual oxidase. ROS quenching by overexpression of superoxide dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H 2 O 2 in minutes. The findings presented reveal that H 2 O 2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism., Competing Interests: AK, PC, LY, DS, TM, KV, AG No competing interests declared, (© 2021, Kizhedathu et al.)

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)-mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ-mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4 -/- Tp53 -/- mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs -/- Tp53 -/- mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ-mediated chromosomal translocation., Competing Interests: The authors declare no competing interest.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G 2 -M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance., (©2021 American Association for Cancer Research.)

Small interfering RNA (siRNA)-based drugs have shown tremendous potential to date in cancer gene therapy. Despite the considerable efforts in siRNA design and manufacturing, unsatisfactory delivery systems persist as a limitation for the application of siRNA-based drugs. In this work, the cholesterol, cell-penetrating peptide conjugate cRGD (R8-cRGD), and polyethylene glycol (PEG) were introduced into low-molecular-weight polyethyleneimine (LMW PEI) to form cRGD-R9-cholesterol-PEI-PEG (RRCPP) nanoparticles with specific targeting and highly penetrating abilities. The enhanced siRNA uptake efficiency of the RRCPP delivery system benefited from R8-cRGD modification. Wee1 is an oncogenic nuclear kinase that can regulate the cell cycle as a crucial G2/M checkpoint. Overexpression of Wee1 in melanoma may lead to a poor prognosis. In the present study, RRCPP nanoparticles were designed for Wee1 siRNA delivery to form an RRCPP/siWee1 complex, which significantly silenced the expression of the WEE1 gene (>60% inhibition) and induced B16 tumor cell apoptosis by abrogating the G2M checkpoint and DNA damage in vitro . Furthermore, the RRCPP/siWee1 complex suppressed B16 tumor growth in a subcutaneous xenograft model (nearly 85% inhibition rate) and lung metastasis (nearly 66% inhibition rate) with ideal in vivo safety. Briefly, our results support the validity of RRCPP as a potential Wee1 siRNA carrier for melanoma gene therapy.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer and a leading cause of cancer-related deaths worldwide. Cinobufotalin (CBF) is extracted from the skin secretion of the giant toad and clinically used for the treatment of liver cancer, but its molecular mechanism of anti-cancer in HCC has not yet been elucidated. Here, we showed CBF effectively promoted cell apoptosis, induced cell cycle G2-M arrest, inhibited cell proliferation and lipogenesis. Consistently, the lipogenesis ability of xenograft examined by 11 C-acetate micro-PET/CT imaging, and the tumor growth rate was notably declined in a centration-dependent manner. The fatty acid profiles showed saturated and mono-unsaturated fatty acid significantly decreased after CBF treatment. Mechanistically, CBF selectively inhibited the expression of SREBP1 and interacted with SREBP1 to prevent it from sterol regulatory elements (SREs), thus inhibiting the expression of lipogenic enzymes. Collectively, our study demonstrates that CBF is a potent native compound that remarkably inhibits HCC lipogenesis and tumorigenesis. CBF may possess this therapeutic potential though interfering with de novo lipid synthesis via SREBP1., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Lung cancer is the most commonly diagnosed malignant cancer in the world. Non-small-cell lung cancer (NSCLC) is the major category of lung cancer. Although effective therapies have been administered, for improving the NSCLC patient's survival, the incident rate is still high. Therefore, searching for a good strategy for preventing NSCLC is urgent. Traditional Chinese medicine (TCM) are brilliant materials for cancer chemoprevention, because of their high biological safety and low cost. Bavachinin, which is an active flavanone of Proralea corylifolia L., possesses anti-inflammation, anti-angiogenesis, and anti-cancer activities. The present study's aim was to evaluate the anti-cancer activity of bavachinin on NSCLC, and its regulating molecular mechanisms. The results exhibited that a dose-dependent decrease in the cell viability and colony formation capacity of three NSCLC cell lines, by bavachinin, were through G2/M cell cycle arrest induction. Meanwhile, the expression of the G2/M cell cycle regulators, such as cyclin B, p-cdc2 Y15 , p-cdc2 T161 , and p-wee1, was suppressed. With the dramatic up-regulation of the cyclin-dependent kinase inhibitor, p21 Waf1/Cip1 , the expression and association of p21 Waf1/Cip1 with the cyclin B/cdc2 complex was observed. Silencing the p21 Waf1/Cip1 expression significantly rescued bavachinin-induced G2/M cell accumulation. Furthermore, the expression of p21 Waf1/Cip1 mRNA was up-regulated in bavachinin-treated NSCLC cells. In addition, MAPK and AKT signaling were activated in bavachinin-added NSCLC cells. Interestingly, bavachinin-induced p21 Waf1/Cip1 expression was repressed after restraint p38 MAPK activation. The inhibition of p38 MAPK activation reversed bavachinin-induced p21 Waf1/Cip1 mRNA expression and G2/M cell cycle arrest. Collectively, bavachinin-induced G2/M cell cycle arrest was through the p38 MAPK-mediated p21 Waf1/Cip1 -dependent signaling pathway in the NSCLC cells.

HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G 2 /M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G 2 cell cycle arrest during infection of CD4 + T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.

A functional p53 protein plays an important role in killing tumor cells. Previous studies showed that chemotherapeutic drug, paclitaxel (PTX), showed anti-tumor activity through inducing G2/M arrest and apoptosis by targeting microtubules in tumor cells. However, PTX was not sensitive to p53-inactivated papillary thyroid carcinoma (PTC) cells by inducing G2/M arrest only. Recombinant adenovirus-p53 (rAd-p53) was used to increase the level of p53, which significantly increased the sensitivity of PTC cells to PTX by inducing S arrest, G2/M arrest and apoptosis. To discuss the anti-tumor mechanism of rAd-p53 + PTX and found p53 activation was necessary for anti-tumor effect of PTX in PTC cells. There was high level of p53 in rAd-p53-treated PTC cells. rAd-p53 + PTX increased the level of p21, p-ATM and γ-H2AX and decreased the level of Cyclin D1/E1, suggesting p53 activated p21 which negatively regulated cyclins to induce S arrest response to DNA damage in PTC cells. rAd-p53 + PTX increased the levels of cleaved-PARP-1, cleaved -Caspase 3, and BAX and decreased the level of BCL-XL, suggesting p53 regulates the expression of BAX/BCL-XL to mediate DNA damage-induced apoptosis in PTC cells. Furthermore, rAd-p53 + PTX showed significant tumor inhibition in TPC-1 xenograft model, with an inhibitory rate of 79.39%. TUNEL assay showed rAd-p53 + PTX induced notable apoptosis in tumor tissues. rAd-p53 showed good sensitization of PTX in vitro and in vivo through inducing DNA damage induced-apoptosis indicated p53-dependent apoptosis was essential for the antitumor effect of PTX in PTC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: This study aimed to confirm the association between developmentally regulated GTP-binding protein 2 (DRG2) expression and docetaxel-induced apoptosis and to determine whether prostate cancer responses to docetaxel treatment differ with DRG2 expression., Materials and Methods: PC3, DU145, and LNCaP prostate cancer cell lines were used. The MTT assay was used to determine cell viability. Western blotting analysis was performed using anti-DRG2 antibodies. Cells were transfected with 50 nmol DRG2 siRNA using an siRNA transfection reagent for DRG2 knockdown. The cell cycle was analyzed by using flow cytometry, and apoptosis was detected by using the Annexin V cell death assay., Results: DRG2 expression differed in each prostate cancer cell line. Docetaxel reduced DRG2 expression in a dose-dependent manner. Upon DRG2 knockdown in prostate cancer cells, an increase in the sub-G1 phase was observed without a change in the G1 or G2/M phases. When 4 nM docetaxel was administered to DRG2 knockdown prostate cancer cell lines, an increase in the sub-G1 phase was observed without increasing the G2/M phase, which was similar to that in DU145 cells before DRG2 knockdown. In PC3 and DU145 cell lines, DRG2 knockdown increased docetaxel-induced Annexin V (+) apoptosis by 8.7 and 2.7 times, respectively., Conclusions: In prostate cancer cells, DRG2 regulates G2/M arrest after docetaxel treatment. In prostate cancer cells with DRG2 knockdown, apoptosis increases without G2/M arrest in response to docetaxel treatment. These results show that inhibition of DRG2 expression can be useful to enhance docetaxel-induced apoptosis despite low-dose administration in castration-resistant prostate cancer., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2021.)

As like in mammalian system, the DNA damage responsive cell cycle checkpoint functions play crucial role for maintenance of genome stability in plants through repairing of damages in DNA and induction of programmed cell death or endoreduplication by extensive regulation of progression of cell cycle. ATM and ATR (ATAXIA-TELANGIECTASIA-MUTATED and -RAD3-RELATED) function as sensor kinases and play key role in the transmission of DNA damage signals to the downstream components of cell cycle regulatory network. The plant-specific NAC domain family transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) plays crucial role in transducing signals from both ATM and ATR in presence of double strand breaks (DSBs) in the genome and found to play crucial role in the regulation of key genes involved in cell cycle progression, DNA damage repair, endoreduplication and programmed cell death. Here we report that Arabidopsis exposed to high salinity shows generation of oxidative stress induced DSBs along with the concomitant induction of endoreduplication, displaying increased cell size and DNA ploidy level without any change in chromosome number. These responses were significantly prominent in SOG1 overexpression line than wild-type Arabidopsis, while sog1 mutant lines showed much compromised induction of endoreduplication under salinity stress. We have found that both ATM-SOG1 and ATR-SOG1 pathways are involved in the salinity mediated induction of endoreduplication. SOG1was found to promote G2-M phase arrest in Arabidopsis under salinity stress by downregulating the expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1, and CYCB1;1, while upregulating the expression of WEE1 kinase, CCS52A and E2Fa, which act as important regulators for induction of endoreduplication. Our results suggest that Arabidopsis undergoes endoreduplicative cycle in response to salinity induced DSBs, showcasing an adaptive response in plants under salinity stress.

Disruption of cell cycle checkpoints has been well established as a hallmark of cancer. In particular, the G1-S transition mediated by the cyclin D-cyclin-dependent kinase 4/6 (CDK4/6) pathway is dysregulated in more than 90% of melanoma cases. Therefore, tumor cells mainly rely on the G2-M checkpoint to halt the cell cycle in order to repair DNA damage. Here, we review the promising method of cell cycle-mediated synthetic lethality for melanoma treatment, which entails exploiting somatically acquired mutations in the G1-S transition with inhibitors of the G2-M transition in order to specifically kill melanoma cells. The idea stems from the theory that melanoma cells lacking G1-S checkpoints are particularly vulnerable to mitotic catastrophe when presented with G2-M checkpoint inhibition in addition to DNA damage, whereas normal cells with intact G1-S checkpoints should theoretically be spared. This review explores the link between cell cycle dysregulation and synthetic lethality in melanoma cells and discusses potential future applications for this treatment.

Spindle assembly abnormal protein 6 homolog (SASS6) is crucial for centriole duplication; however, the role of SASS6 in the proliferation of cancer cells remains unclear. In the present study, the expression and functional role of SASS6 in triple negative breast cancer (TNBC) was assessed. Immunohistochemical staining was performed using an anti‑SASS6 antibody in TNBC and normal tissues. Lentivirus‑mediated RNA interference was used to knockdown SASS6 in MDA‑MB‑231 TNBC cells. Cell viability was determined using an MTT assay, and cell cycle distribution and apoptosis were measured using flow cytometry. Additionally, PathScan intracellular signaling arrays were used to detect the presence of intracellular signaling molecules. The results revealed that SASS6 expression was increased in TNBC tissues compared with the control tissue. Moreover, SASS6 knockdown significantly suppressed the growth of MDA‑MB‑231 cells. MDA‑MB‑231 cell cycle progression was arrested at the G2/M phase and cyclin dependent kinase 1 (CDK1), cyclin B1 and PCNA expression in MDA‑MB‑231 cells was decreased following SASS6 knockdown. Furthermore, the phosphorylation of STAT3, BAD and rpS6 was reduced following SASS6 knockdown. A strong correlation between SASS6 and CDK1 expression was observed in TNBC tissues based on immunohistochemical staining analysis (R=0.989; P<0.001). In conclusion, the present study revealed the crucial role of SASS6 in promoting MDA‑MB‑231 cell growth, regulating cell cycle progression and its ability to downregulate the CDK1/cyclin B1 signaling pathway, thus highlighting the potential of SASS6 as a therapeutic target for treatment of TNBC, and merits further investigation in animal models or in preclinical and clinical studies.

The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer-polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14's nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA-binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA-DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer-polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent studies have revealed that long noncoding RNAs (lncRNAs) are closely associated with colorectal cancer (CRC); however, the role of the lncRNA RPLP0P2 in CRC remains largely unknown. In the present study, RNA expression profiles of CRC were collected from The Cancer Genome Atlas database and the prognosis of CRC with respect to RPLP0P2 was assessed. Subsequently, RPLP0P2 expression was knocked down in the human CRC cell line RKO using a short hairpin RNA (shRNA) lentivirus, and the biological behaviors of the cells, such as proliferation, migration, cell cycle progression and apoptosis, were examined. The results demonstrated that the expression levels of RPLP0P2 were higher in CRC tissue compared with those in normal tissue, and RPLP0P2 was associated with prognosis. RPLP0P2 knockdown significantly decreased cell colony formation, migration and invasion, and arrested CRC cells in the S phase to G2/M phase transition. Furthermore, apoptosis was significantly increased in CRC cells infected with the RPLP0P2 shRNA lentivirus compared with in the control group. In conclusion, RPLP0P2 may promote proliferation, invasion and migration, and inhibit apoptosis of CRC cells, suggesting that RPLP0P2 may function as an oncogene in CRC.

Purpose: Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes., Methods: We analyzed in vitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated in vivo in a heterotopic mouse xenograft model., Results: Our in vitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 μs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. In vivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation in vivo to enable complete inhibition of tumor growth., Conclusions: Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Forkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported., Methods: Transcriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database., Results: FOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-O FOXD1null . In vivo, xenografted 786-O FOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-O FOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-O FOXD1null , and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation., Conclusions: We show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

Pulmonary atresia with ventricular septal defect (PA/VSD) is a rare congenital heart disease (CHD) characterized by a lack of luminal continuity and blood flow from either the right ventricle or the pulmonary artery, together with VSDs. The prevalence of PA/VSD is about 0.2% of live births and approximately 2% of CHDs. PA/VSD is similar to tetralogy of Fallot (TOF) in terms of structural and pathological characteristics. The pathogenesis of these two CHDs remains incompletely understood. It was previously reported that N-myc downstream-regulated gene (NDRG)4 is required for myocyte proliferation during early cardiac development. In the present study, we enrolled 80 unrelated patients with PA/VSD or TOF and identified a probably damaging variant p.T256M of NDRG4. The p.T256M variant impaired the proliferation ability of human cardiac myocytes (hCM). Furthermore, the p.T256M variant resulted in G1 and G2 arrest of hCM, followed by an increase in p27 and caspase-9 expression. Our results provide evidence that the p.T256M variant in NDRG4 is a pathogenic variant associated with impaired hCM proliferation and cell-cycle arrest and likely contributes towards the pathogenesis of PA/VSD and TOF., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

Renal fibrosis is the common feature of all progressive kidney diseases and exerts great burden on public health worldwide. The maladaptive repair mechanism of tubular epithelial cells, an important mediator of renal fibrogenesis, manifests with partial epithelial-mesenchymal transition (EMT) and cell cycle arrest. The aim of this study is to investigate the possible correlation between partial EMT and cell cycle arrest, and elucidate the underlying mechanism. We examined human kidney allograft samples with interstitial fibrosis and three mice renal fibrosis models, unilateral ureter obstruction (UUO), ischemia-reperfusion injury, and Adriamycin nephropathy. The partial EMT process and p53-p21 axis were elevated in both human allograft with interstitial fibrosis, as well as three mice renal fibrosis models, and showed a time-dependent increase as fibrosis progressed in the UUO model. Snai1 controlled the partial EMT process, and led to parallel changes in renal fibrosis, G2/M arrest, and inflammation. p53-p21 axis arrested cell cycle at G2/M, and prompted partial EMT and fibrosis together with inflammation. NF-κB inhibitor Bay11-7082 disrupted the reciprocal loop between Snai1-induced partial EMT and p53-p21-mediated G2/M arrest. We demonstrated the reciprocal loop between partial EMT and G2/M arrest of TECs during renal fibrogenesis and revealed NF-κB-mediated inflammatory response as the underlying mechanism. This study suggests that targeting NF-κB might be a plausible therapeutic strategy to disrupt the reciprocal loop between partial EMT and G2/M arrest, therefore alleviating renal fibrosis.

Cancer is the second leading cause of death globally, where nearly 1 in 6 deaths is due to cancer, with 70% of all deaths from cancer occur in low- and middle-income countries. The overall lifetime risk of developing colorectal cancer is 1 in 22 in men and 1 in 24 in women. In this work, we aimed to evaluate the role of temozolomide (TMZ) in controlling colon cancer cells (CRC) via regulating the miRnome. For this purpose, CRC cells (CaCo-2) were treated with 50 µM of TMZ for 48 h. Cell count using trypan test and cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were carried out, and the obtained results indicated a significant decrease in cell count (p = 0.029), and in the cell viability (p = 0.0019). Cell cycle analysis was performed using flow cytometer, and results showed that TMZ arrested CRC cells at G2/M phase. A total of 84 miRNAs were profiled using real time PCR, and the results indicated that TMZ treatment upregulated 15 of 84 miRNAs panel profiled and downregulated the rest. The TMZ-upregulated/downregulated miRNAs were predicted to interact with many epigenetic-related proteins i.e., DNMTs, EZH2, and SUV31H1. This study shed some light on the role of TMZ in regulating the miRnome of CRC and hence in different types of cancers., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mouse embryonic stem cells (mESCs) are pluripotent cells that possess the ability to self-renew and differentiate into three germ layers. Owing to these characteristics, mESCs act as important models for stem cell research and are being used in many clinical applications. Among the many cathepsins, cathepsin A (Ctsa), a serine protease, affects the function and properties of stem cells. However, studies on the role of Ctsa in stem cells are limited. Here, we observed a significant increase in Ctsa expression during mESC differentiation at protein levels. Furthermore, we established Ctsa knockdown mESCs. Ctsa knockdown led to Erk1/2 phosphorylation, which in turn inhibited the pluripotency of mESCs and induced G2/M cell cycle arrest to inhibit mESC proliferation. The knockdown also induced abnormal differentiation in mESCs and aberrant expression of differentiation markers. Furthermore, we identified inhibition of teratoma formation in nude mice. Our results suggested that Ctsa affects mESC pluripotency, proliferation, cell cycle and differentiation, and highlighted the potential of Ctsa to act as a core factor that can regulate various mESC properties. SIGNIFICANCE OF THE STUDY: Our results indicate that cathepsin A (Ctsa) affects the properties of mESCs. Inhibition of Ctsa resulted in a decrease in the pluripotency of mouse embryonic stem cells (mESCs). Further, Ctsa suppression resulted in decreased proliferation via cell cycle arrest. Moreover, Ctsa inhibition reduced differentiation abilities and formation of teratoma in mESCs. Our results demonstrated that Ctsa is an important factor controlling mESC abilities., (© 2020 John Wiley & Sons Ltd.)

Purpose: Insulin like growth factor receptor 1 (IGF-1R) has been documented to play a key role in radiation response, thereby offering an attractive drug target to enhance tumor sensitivity to radiotherapy. Here, we investigated wether knockdown of IGF-1R can sensitize colorectal cancer (CRC) cell lines to radiation., Material and Methods: Human colon carcinoma SW480 and HT-29 cells were transfected with specific small interference RNA (siRNA) to mediate IGF-1R depletion. The expression of IGF-1R mRNA and protein among transfected and untransfected cells was detected by Western blot analysis. Changes in cell proliferation and radiosensitivity were evaluated by the clonogenic survival assay. NVP-ADW742, an IGF-1R inhibitor, in combination with radiation was studied. RAD51, a measure for homologous recombination repair, and 53BP1, a maker for non-homologous end-joining (NHEJ), were determined by immunofluorescence for double-strand breaks (DSB) repair pathways. Cell cycle was also examined in the IGF-1R knockdown and IGF-1R-inhibited cells., Results: CRC cell lines were selectively sensitized to radiation after siRNA-mediated IGF-1R depletion. NVP-ADW742 efficiently increases cancer cell response to radiation. Furthermore, initial formation of RAD51 foci after IR, and 53BP1 foci were significantly reduced in IGF-1R-depleted or with IGF-1R Inhibitor CRC cell lines. Lastly, IGF-1R-depleted or with IGF-1R Inhibitor caused more G2 phase cell arrest., Conclusion: Our findings demonstrate that depletion of IGF-1R lead to an increase in radiosensitivity in CRC.

Despite advances in the diagnosis and treatment of the central nervous system malignancy glioma, overall survival remains poor. Cytoskeleton-associated protein 2-like ( CKAP2L ), which plays key roles in neural progenitor cell division, has also been linked to poor prognosis in lung cancer. In the present study, we investigated the role of CKAP2L in glioma. From bioinformatics analyses of datasets from The Cancer Gene Atlas and the Chinese Glioma Genome Atlas, we found that CKAP2L expression correlates with tumor grade and overall survival. Gene set enrichment analysis (GSEA) showed that MITOTIC&#95;SPINDLE, G2M&#95;CHECKPOINT, and E2F&#95;TARGETS are crucially enriched phenotypes associated with high CKAP2L expression. Using U87MG, U118MG, and LNZ308 human glioma cells, we confirmed that CKAP2L knockdown with si CKAP2L inhibits glioma cell proliferation, migration, invasion, and epithelial-mesenchymal transition. Interestingly, CKAP2L knockdown also induced cell cycle arrest at G2/M phase, which is consistent with the GSEA finding. Finally, we observed that CKAP2L knockdown led to significant increases in miR-4496. Treating cells with exogenous miR-4496 mimicked the effect of CKAP2L knockdown, and the effects of CKAP2L knockdown could be suppressed by miR-4496 inhibition. These findings suggest that CKAP2L is a vital regulator of miR-4496 activity and that CKAP2L is a potentially useful prognostic marker in glioma.

CFL2, a skeletal muscle-specific member of the actin depolymerizing factor/cofilin protein family, is known to be involved in the regulation of actin filament dynamics. Although the impact of CFL2 has been studied in human myopathy, its functional contribution to myogenic differentiation, in terms of its effects on cell proliferation, cell cycle, and myogenic factor modulation, remains largely unknown. Here, we report that CFL2 is required for the myogenic differentiation of C2C12 myoblasts by regulating proliferation and myogenic transcription factors expressions. CFL2 expression was induced during myogenic progression, and its knockdown by siRNA in myoblasts enhanced phalloidin staining, indicating increased filamentous actin formation. Interestingly, CFL2 depletion stimulated cell proliferation and induced a cell cycle shift from G0/G1 to G2/M phases, which are known to inhibit progenitor cell differentiation. CFL2 knockdown markedly downregulated the protein expressions of myogenic transcription factors (MyoD, MyoG, and MEF2C) and thereby impaired the differentiation and myotube formation of C2C12 myoblasts. Collectively, this study highlights the roles played by CFL2 on cell cycle progression and proliferation and suggests a novel regulatory mechanism of myogenic differentiation mediated by CFL2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ . Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

Background: Mutations in one allele of the TP53 gene in early stages are frequently followed by the loss of the remaining wild-type p53 (wtp53) allele (p53LOH) during tumor progression. Despite the strong notion of p53LOH as a critical step in tumor progression, its oncogenic outcomes that facilitate the selective pressure for p53LOH occurrence were not elucidated., Methods: Using MMTV;ErbB2 mouse model of breast cancer carrying heterozygous R172H p53 mutation, we identified a novel gain-of-function (GOF) activity of mutant p53 (mutp53): the exacerbated loss of wtp53 allele in response to γ-irradiation., Results: As consequences of p53LOH in mutp53 heterozygous cells, we observed profound stabilization of mutp53 protein, the loss of p21 expression, the abrogation of G2/M checkpoint, chromosomal instability, centrosome amplification, and transcriptional upregulation of mitotic kinase Nek2 (a member of Never in Mitosis (NIMA) Kinases family) involved in the regulation of centrosome function. To avoid the mitotic catastrophe in the absence of G2/M checkpoint, cells with centrosome amplification adapt Nek2-mediated centrosomes clustering as pro-survival mutp53 GOF mechanism enabling unrestricted proliferation and clonal expansion of cells with p53LOH. Thus, the clonal dominance of mutp53 cells with p53LOH may represent the mechanism of irradiation-induced p53LOH. We show that pharmacological and genetic ablation of Nek2 decreases centrosome clustering and viability of specifically mutp53 cells with p53LOH., Conclusion: In a heterogeneous tumor population, Nek2 inhibition may alter the selective pressure for p53LOH by contraction of the mutp53 population with p53LOH, thus, preventing the outgrowth of genetically unstable, more aggressive cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Ubiquitin‑specific protease 12 (USP12) is specifically upregulated in the tumor tissues of patients with HCC compared with the corresponding adjacent normal tissues. However, the relationship between USP12 and the growth of HCCs is not fully understood. In the present study, USP12 was knocked down in HCC cell lines to investigate its effects on proliferation and apoptosis. The results showed that USP12‑knockdown could inhibit the proliferation and promote apoptosis in HCC cell lines. Flow cytometry analysis also showed that USP12 could induce cell cycle arrest at the G2/M stage. In vivo experiments showed that USP12‑knockdown could suppress tumor growth in mice, and immuno‑blotting revealed that USP12 could induce G2/M arrest through the cyclin dependent kinase 1/cyclinB1 axis, and trigger apoptosis via the p38/mitogen‑activated protein kinase pathway. These data strongly indicate that USP12 is a potential target for the treatment of HCC.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and its long‑term survival rate has stagnated in the past decades. Previous studies have shown that tumors in the G2/M phase are more sensitive to radiotherapy. The proto‑oncogene c‑myc is a transformed member of the myc family and c‑myc‑interacting zinc finger protein‑1 (Miz‑1) is a poly‑Cys2His2 zinc finger (ZF) activator of cell cycle regulator genes, such as the cyclin‑dependent kinase inhibitor p21. C‑myc can repress the expression of p21 by binding to Miz‑1 and abolishing the interaction between Miz‑1 and its co‑activators, which induces G2/M phase arrest. Therefore, the present study investigated the radiosensitizing effects of the c‑myc gene and the sensitizing apoptosis pathway, aiming to identify a more effective combination radiotherapy treatment for osteosarcoma. The present study demonstrated that the c‑myc gene was overexpressed in osteosarcoma cells compared to osteoblasts. Following inhibition of c‑myc gene expression in osteosarcoma cells, tumor proliferation was significantly hindered after inducing G2/M phase arrest via regulating G2/M phase‑associated proteins. Additionally, it was revealed that inhibiting c‑myc gene expression combined with radiotherapy could significantly increase the apoptosis rate of osteosarcoma cells via the mitochondrial signaling pathway. In summary, the present study verified the radiosensitizing effects of c‑myc gene knockdown‑induced G2/M phase arrest, which was achieved by intrinsic stimuli through the mitochondrial signaling pathway.