ABSTRACT
DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.
Subject(s)
DNA Topoisomerases, Type II , Heterochromatin , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Heterochromatin/metabolism , Animals , Topoisomerase II Inhibitors/pharmacology , Repetitive Sequences, Nucleic Acid/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Replication , DNA, Superhelical/metabolism , DNA, Superhelical/chemistry , Humans , Mice , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA/metabolism , DNA/chemistry , InterphaseABSTRACT
Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.
Subject(s)
Androgens , Prostatic Neoplasms , R-Loop Structures , RNA Polymerase II , Receptors, Androgen , Humans , Androgens/metabolism , Receptors, Androgen/metabolism , Receptors, Androgen/genetics , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Transcription, Genetic , DNA Breaks, Double-Stranded , Transcription Initiation Site , Gene Expression Regulation, Neoplastic , Protein Binding , Transcriptional ActivationABSTRACT
Trichomes are specialized epidermal structures that protect plants from biotic and abiotic stresses by synthesizing, storing, and secreting defensive compounds. This study investigates the role of the Gossypium arboreum DNA topoisomerase VI subunit B gene (GaTOP6B) in trichome development and branching. Sequence alignment revealed a high similarity between GaTOP6B and AtTOP6B, suggesting a conserved function in trichome regulation. Although AtTOP6B acts as a positive regulator of trichome development, functional analyses showed contrasting effects: Virus-induced gene silencing (VIGS) of GaTOP6B in cotton increased trichome density, while its overexpression in Arabidopsis decreased trichome density but enhanced branching. This demonstrates that GaTOP6B negatively regulates trichome number, indicating species-specific roles in trichome initiation and branching between cotton and Arabidopsis. Overexpression of the GaTOP6B promotes jasmonic acid synthesis, which in turn inhibits the G1/S or G2/M transitions, stalling the cell cycle. On the other hand, it suppresses brassinolide synthesis and signaling while promoting cytokinin degradation, further inhibiting mitosis. These hormonal interactions facilitate the transition of cells from the mitotic cycle to the endoreduplication cycle. As the level of endoreduplication increases, trichomes develop an increased number of branches. These findings highlight GaTOP6B's critical role as a regulator of trichome development, providing new genetic targets for improving cotton varieties in terms of enhanced adaptability and resilience.
Subject(s)
Arabidopsis , Cyclopentanes , Endoreduplication , Gene Expression Regulation, Plant , Gossypium , Oxylipins , Plant Proteins , Trichomes , Trichomes/genetics , Trichomes/growth & development , Trichomes/metabolism , Gossypium/genetics , Gossypium/growth & development , Gossypium/metabolism , Cyclopentanes/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Oxylipins/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Endoreduplication/genetics , Brassinosteroids/metabolism , Plants, Genetically Modified , Genes, Plant , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Steroids, HeterocyclicABSTRACT
The discovery of novel oncotargets for glioma is of immense significance. We here explored the expression patterns, biological functions, and underlying mechanisms associated with ORC6 (origin recognition complex 6) in glioma. Through the bioinformatics analyses, we found a significant increase in ORC6 expression within human glioma tissues, correlating with poorer overall survival, higher tumor grade, and wild-type isocitrate dehydrogenase status. Additionally, ORC6 overexpression is detected in glioma tissues obtained from locally-treated patients and across various primary/established glioma cells. Further bioinformatics scrutiny revealed that genes co-expressed with ORC6 are enriched in multiple signaling cascades linked to cancer. In primary and immortalized (A172) glioma cells, depleting ORC6 using specific shRNA or Cas9-sgRNA knockout (KO) significantly decreased cell viability and proliferation, disrupted cell cycle progression and mobility, and triggered apoptosis. Conversely, enhancing ORC6 expression via a lentiviral construct augmented malignant behaviors in human glioma cells. ORC6 emerged as a crucial regulator for the expression of key oncogenic genes, including Cyclin A2, Cyclin B2, and DNA topoisomerase II (TOP2A), within glioma cells. Silencing or KO of ORC6 reduced the mRNA and protein levels of these genes, while overexpression of ORC6 increased their expression in primary glioma cells. Bioinformatics analyses further identified RBPJ as a potential transcription factor of ORC6. RBPJ shRNA decreased ORC6 expression in primary glioma cells, while its overexpression increased it. Additionally, significantly enhanced binding between the RBPJ protein and the proposed ORC6 promoter region was detected in glioma tissues and cells. In vivo experiments demonstrated a significant reduction in the growth of patient-derived glioma xenografts in the mouse brain subsequent to ORC6 KO. ORC6 depletion, inhibited proliferation, decreased expression of Cyclin A2/B2/TOP2A, and increased apoptosis were detected within these ORC6 KO intracranial glioma xenografts. Altogether, RBPJ-driven ORC6 overexpression promotes glioma cell growth, underscoring its significance as a promising therapeutic target.
Subject(s)
Cell Proliferation , Gene Expression Regulation, Neoplastic , Glioma , Origin Recognition Complex , Animals , Humans , Male , Mice , Apoptosis/genetics , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cyclin A2/metabolism , Cyclin A2/genetics , Cyclin B2/metabolism , Cyclin B2/genetics , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Glioma/genetics , Glioma/pathology , Glioma/metabolism , Mice, Nude , Origin Recognition Complex/metabolism , Origin Recognition Complex/geneticsABSTRACT
Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Chromosome Segregation , DNA Topoisomerases, Type II , Spermatogenesis , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Spermatogenesis/genetics , Male , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Meiosis , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/genetics , MutationABSTRACT
DNA Topoisomerase IIα (Top2A) is a nuclear enzyme that is a cancer drug target, and there is interest in identifying novel sites on the enzyme to inhibit cancer cells more selectively and to reduce off-target toxicity. The C-terminal domain (CTD) is one potential target, but it is an intrinsically disordered domain, which prevents structural analysis. Therefore, we set out to analyze the sequence of Top2A from 105 species using bioinformatic analysis, including the PSICalc algorithm, Shannon entropy analysis, and other approaches. Our results demonstrate that large (10th-order) interdependent clusters are found including non-proximal positions across the major domains of Top2A. Further, CTD-specific clusters of the third, fourth, and fifth order, including positions that had been previously analyzed via mutation and biochemical assays, were identified. Some of these clusters coincided with positions that, when mutated, either increased or decreased relaxation activity. Finally, sites of low Shannon entropy (i.e., low variation in amino acids at a given site) were identified and mapped as key positions in the CTD. Included in the low-entropy sites are phosphorylation sites and charged positions. Together, these results help to build a clearer picture of the critical positions in the CTD and provide potential sites/regions for further analysis.
Subject(s)
Computational Biology , DNA Topoisomerases, Type II , Protein Domains , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/chemistry , Computational Biology/methods , Humans , Entropy , Amino Acid Sequence , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/chemistry , PhosphorylationABSTRACT
BACKGROUND/AIM: Hyperthermia represents an adjuvant local anticancer strategy which relies on the increase of temperature beyond the physiological level. In this study, we investigated the anticancer potential of Fe3O4 and Fe3O4core Aushell nanoparticles as hyperthermic agents in terms of cytotoxicity and studied the expression of cellular markers of proliferation (changes in mRNA levels via real-time polymerase chain reaction). MATERIALS AND METHODS: The human breast cancer cell line SK-BR-1 was incubated with either Fe3O4 or Fe3O4core Aushell nanoparticles stabilized with tryptophan, prior to hyperthermia treatment. The normal HEK293 cell line was used as a control. Toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to estimate possible toxic effects of the tested nanoparticles. After RNA extraction and cDNA synthesis, mRNA expression of three indicators of proliferation, namely marker of proliferation Ki-67, DNA topoisomerase II alpha (TOP2A) and TPX2 microtubule nucleation factor (TPX2), was investigated. RESULTS: At each concentration tested, Fe3O4core Aushell nanoparticles showed greater toxicity compared to Fe3O4, while SK-BR-3 cells were more susceptible to their cytotoxic effects compared to the HEK293 cell line. The expression of Ki-67, TOP2A and TPX2 was reduced in SK-BR-3 cells by both Fe3O4 or Fe3O4core Aushell nanoparticles compared to untreated cells, while the only observed change in HEK293 cells was the up-regulation of TOP2A. CONCLUSION: Both Fe3O4core Aushell and Fe3O4 NPs exhibit increased cytotoxicity to the cancer cell line tested (SK-BR-3) compared to HEK293 cells. The down-regulation in SK-BR-3 cells of the three proliferative markers studied, Ki-67, TOP2A and TPX2, after incubation with NPs suggests that cells that survived thermal destruction were not actively proliferating.
Subject(s)
Breast Neoplasms , Cell Cycle Proteins , Cell Proliferation , DNA Topoisomerases, Type II , Hyperthermia, Induced , Ki-67 Antigen , Microtubule-Associated Proteins , Poly-ADP-Ribose Binding Proteins , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/drug therapy , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Cell Proliferation/drug effects , Hyperthermia, Induced/methods , Ki-67 Antigen/metabolism , Ki-67 Antigen/genetics , Cell Line, Tumor , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Female , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , HEK293 Cells , Gene Expression Regulation, Neoplastic/drug effects , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/geneticsABSTRACT
DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa; TOP2α/90), which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (AATAAAâACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance.
Subject(s)
DNA Topoisomerases, Type II , Drug Resistance, Neoplasm , Etoposide , Introns , Polyadenylation , Humans , Etoposide/pharmacology , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , K562 Cells , Polyadenylation/drug effects , Polyadenylation/genetics , Introns/genetics , CRISPR-Cas SystemsABSTRACT
Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond-scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N and C termini of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point toward specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.
Subject(s)
DNA Topoisomerases, Type II , Molecular Dynamics Simulation , Allosteric Regulation , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Protein Domains , Models, MolecularABSTRACT
DNA topoisomerase II alpha (TOP2A) expression, gene alterations, and enzyme activity have been studied in various malignant tumors. Abnormal elevation of TOP2A expression is considered to be related to the development of non-small cell lung cancer (NSCLC). However, its association with tumor metastasis and its mode of action remains unclear. Bioinformatics, real-time quantitative PCR, immunohistochemistry and immunoblotting were used to detect TOP2A expression in NSCLC tissues and cells. Cell migration and invasion assays as well as cytoskeletal staining were performed to analyze the effects of TOP2A on the motility, migration and invasion ability of NSCLC cells. Cell cycle and apoptosis assays were used to verify the effects of TOP2A on apoptosis as well as cycle distribution in NSCLC. TOP2A expression was considerably upregulated in NSCLC and significantly correlated with tumor metastasis and the occurrence of epithelial-mesenchymal transition (EMT) in NSCLC. Additionally, by interacting with the classical ligand Wnt3a, TOP2A may trigger the canonical Wnt signaling pathway in NSCLC. These observations suggest that TOP2A promotes EMT in NSCLC by activating the Wnt/ß-catenin signaling pathway and positively regulates malignant events in NSCLC, in addition to its significant association with tumor metastasis. TOP2A promotes the metastasis of NSCLC by stimulating the canonical Wnt signaling pathway and inducing EMT. This study further elucidates the mechanism of action of TOP2A, suggesting that it might be a potential therapeutic target for anti-metastatic therapy.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Cell Movement , DNA Topoisomerases, Type II , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Lung Neoplasms , Poly-ADP-Ribose Binding Proteins , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Epithelial-Mesenchymal Transition/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Cell Movement/genetics , Cell Line, Tumor , Neoplasm Metastasis , Wnt Signaling Pathway , Apoptosis , Male , Female , Middle Aged , Wnt3A Protein/metabolism , Wnt3A Protein/geneticsABSTRACT
BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa, with a gradually increasing incidence. Therefore, it is necessary to actively seek targets for the treatment of UC. METHODS: Common differentially expressed genes (DEGs) were screened from two microarray data sets related to UC. Protein-protein interaction network was constructed to find the hub genes. The UC mouse model and cell model were induced by dextran sulfate sodium (DSS). The pathological changes of colon tissue were observed by hematoxylin-eosin staining. Immunohistochemistry and immunofluorescence were performed to detect the expressions of Ki67 and Claudin-1. The performance of mice was observed by disease activity index (DAI). The effect of TOP2A on proliferation, inflammation, oxidative stress, and interleukin-17 (IL-17) signaling pathway in UC model was measured by cell counting kit-8, enzyme-linked immunosorbent assay, and western blot. RESULTS: Through bioinformatics analysis, 295 common DEGs were screened, and the hub gene TOP2A was selected. In UC model, there was obvious inflammatory cell infiltration in the colon and less goblet cells, while si-TOP2A lessened it. More Ki67 positive cells and less Claudin-1 positive cells were observed in UC model mice. Furthermore, knockdown of TOP2A increased the body weight and colon length of UC mice, while the DAI was decreased. Through in vivo and in vitro experiments, knockdown of TOP2A also inhibited inflammation and IL-17 signaling pathway, and promoted proliferation in DSS-induced NCM460 cells. CONCLUSION: Knockdown of TOP2A alleviated the progression of UC by suppressing inflammation and inhibited IL-17 signaling pathway.
Subject(s)
Colitis, Ulcerative , DNA Topoisomerases, Type II , Disease Models, Animal , Disease Progression , Interleukin-17 , Poly-ADP-Ribose Binding Proteins , Signal Transduction , Animals , Humans , Male , Mice , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/genetics , Colitis, Ulcerative/metabolism , Dextran Sulfate , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , Gene Knockdown Techniques , Interleukin-17/metabolism , Interleukin-17/genetics , Protein Interaction MapsABSTRACT
The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.
Subject(s)
Chromosomes , DNA Topoisomerases, Type II , DNA Topoisomerases, Type II/genetics , Chromosomes/genetics , DNA/genetics , Mitosis/geneticsABSTRACT
BACKGROUND: Patients with breast cancer (BC) at advanced stages have poor outcomes because of high rate of recurrence and metastasis. Biomarkers for predicting prognosis remain to be explored. This study aimed to evaluate the relationships between circulating tumor cells (CTCs) and outcomes of BC patients. PATIENTS AND METHODS: A total of 50 female were enrolled in this study. Their diagnoses were determined by clinical characteristics, image data, and clinical pathology. CTC subtypes and TOP2A gene expression on CTCs were detected by CanPatrol™ technology and triple color in situ RNA hybridization (RNA-ISH), which divided into epithelial CTCs (eCTCs), mesenchymal CTCs (MCTCs), and hybrid CTCs (HCTCs) based on their surface markers. Hormone receptor, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, was measured by immunohistochemistry (IHC) method before treatment. The risk factors for predicting recurrence and metastasis were calculated by COX risk regression model. The progression-free survival (PFS) of patients was determined using Kaplan-Meier survival curve. RESULTS: The patients with a large tumor size (≥ 3 cm) and advanced tumor node metastasis (TNM) stages had high total CTCs (TCTCs) (P < 0.05). These patients also had high TOP2A expression level. COX risk regression analysis indicated that TOP2A expression levels in TCTCs, ER + , HER-2 + , and TNM stages were critical risk factors for recurrence and metastasis of patients (P < 0.05). The PFS of patients with ≥ 5 TCTCs, ≥ 3 HCTCs, and positive TOP2A expression in ≥ 3 TCTCs was significantly longer than that in patient with < 5 TCTCs, < 3 HCTCs, and TOP2A expression in < 3 TCTCs (P < 0.05). In contrast, the PFS of patients with positive hormone receptors (ER + , PR + , HER-2 +) also was dramatically lived longer than that in patients with negative hormone receptor expression. CONCLUSIONS: High TCTC, HCTCs, and positive TOP2A gene expression on CTCs were critical biomarkers for predicting outcomes of BC patients. Positive hormone receptor expression in BC patients has significant favor PFS.
Subject(s)
Biomarkers, Tumor , Breast Neoplasms , DNA Topoisomerases, Type II , Drug Resistance, Neoplasm , Neoplastic Cells, Circulating , Humans , Female , Neoplastic Cells, Circulating/metabolism , Neoplastic Cells, Circulating/pathology , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Middle Aged , Drug Resistance, Neoplasm/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Adult , Aged , Receptor, ErbB-2/metabolism , Prognosis , Receptors, Estrogen/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/genetics , Receptors, Progesterone/metabolism , Gene Expression Regulation, Neoplastic , Progression-Free Survival , Kaplan-Meier EstimateABSTRACT
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Subject(s)
Chromosomes , DNA Topoisomerases, Type II , DNA Topoisomerases, Type II/genetics , Chromosomes/genetics , Mitosis/genetics , Interphase/genetics , PolymersABSTRACT
Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract. Topoisomerase II α (TOP2A) is commonly regarded as an oncogene that is associated with malignant disease progression in a variety of cancers, its mechanistic functions in OC have yet to be firmly established. We explored the role of TOP2A in OC through online databases, clinical samples, in vitro and in vivo experiments. And initial analyses of public databases revealed high OC-related TOP2A expression in patient samples that was related to poorer prognosis. This was confirmed by clinical samples in which TOP2A expression was elevated in OC relative to healthy tissue. Kaplan-Meier analyses further suggested that higher TOP2A expression levels were correlated with worse prognosis in OC patients. In vitro, TOP2A knockdown resulted in the inhibition of OC cell proliferation, with cells entering G1 phase arrest and undergoing consequent apoptotic death. In rescue assays, TOP2A was confirmed to regulate cell proliferation and cell cycle through AKT/mTOR pathway activity. Mouse model experiments further affirmed the key role that TOP2A plays as a driver of OC cell proliferation. These data provide strong evidence supporting TOP2A as an oncogenic mediator and prognostic biomarker related to OC progression and poor outcomes. At the mechanistic level, TOP2A can control tumor cell growth via AKT/mTOR pathway modulation. These preliminary results provide a foundation for future research seeking to explore the utility of TOP2A inhibitor-based combination treatment regimens in platinum-resistant recurrent OC patients.
Subject(s)
Ovarian Neoplasms , Proto-Oncogene Proteins c-akt , Animals , Female , Humans , Mice , Carcinoma, Ovarian Epithelial , Cell Proliferation , DNA Topoisomerases, Type II/genetics , Ovarian Neoplasms/genetics , TOR Serine-Threonine KinasesABSTRACT
CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.
Subject(s)
CCCTC-Binding Factor , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type II , DNA , Nucleic Acid Conformation , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/chemistry , Humans , CCCTC-Binding Factor/metabolism , CCCTC-Binding Factor/genetics , Binding Sites , DNA/metabolism , DNA/chemistry , DNA/genetics , Protein Binding , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/chemistry , Cell LineABSTRACT
DNA topoisomerase IIß (TOP2ß/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2ß/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2ß/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2ß/180, a premature stop codon was generated at the TOP2ß/180 gene exon 19/intron 19 boundary (AGAA//GTAAâATAG//GTAA) in parental K562 cells (which contain four TOP2ß/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2ß/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2ß/180 gene-edited clones, with one or all four TOP2ß/180 alleles mutated, revealed partial or complete loss of TOP2ß mRNA/protein, respectively. The loss of TOP2ß/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2ß/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2ß/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2ß/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.
Subject(s)
Antineoplastic Agents , Leukemia , Humans , Etoposide/pharmacology , K562 Cells , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Mitoxantrone , CRISPR-Cas Systems/genetics , Codon, Nonsense , Antineoplastic Agents/pharmacology , DNA , PhenotypeABSTRACT
PURPOSE: Previous studies have reported the benefit of dual HER2-targeting combined to neoadjuvant chemotherapy in HER2-amplified breast cancer (HER2 + BC). Moreover, besides the cardiac toxicity following their association to Trastuzumab, anthracyclines chemotherapy may not profit all patients. The NeoTOP study was designed to evaluate the complementary action of Trastuzumab and Pertuzumab, and the relevance of an anthracycline-based regimen according to TOP2A amplification status. METHODS: Open-label, multicentre, phase II study. Eligible patients were aged ≥ 18 with untreated, operable, histologically confirmed HER2 + BC. After centralized review of TOP2A status, TOP2A-amplified (TOP2A+) patients received FEC100 for 3 cycles then 3 cycles of Trastuzumab (8 mg/kg then 6 mg/kg), Pertuzumab (840 mg/kg then 420 mg/kg), and Docetaxel (75mg/m2 then 100mg/m2). TOP2A-not amplified (TOP2A-) patients received 6 cycles of Docetaxel (75mg/m2) and Carboplatin (target AUC 6 mg/ml/min) plus Trastuzumab and Pertuzumab. Primary endpoint was pathological Complete Response (pCR) using Chevallier's classification. Secondary endpoints included pCR (Sataloff), Progression-Free Survival (PFS), Overall Survival (OS), and toxicity. RESULTS: Out of 74 patients, 41 and 33 were allocated to the TOP2A + and TOP2A- groups respectively. pCR rates (Chevallier) were 74.4% (95%CI: 58.9-85.4) vs. 71.9% (95%CI: 54.6-84.4) in the TOP2A + vs. TOP2A- groups. pCR rates (Sataloff), 5-year PFS and OS were 70.6% (95%CI: 53.8-83.2) vs. 61.5% (95%CI: 42.5-77.6), 82.4% (95%CI: 62.2-93.6) vs. 100% (95%CI: 74.1-100), and 90% (95%CI: 69.8-98.3) vs. 100% (95%CI: 74.1-100). Toxicity profile was consistent with previous reports. CONCLUSION: Our results showed high pCR rates with Trastuzumab and Pertuzumab associated to chemotherapy. They were similar in TOP2A + and TOP2A- groups and the current role of neoadjuvant anthracycline-based chemotherapy remains questioned. TRIAL REGISTRATION NUMBER: NCT02339532 (registered on 14/12/14).
Subject(s)
Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols , Breast Neoplasms , Carboplatin , DNA Topoisomerases, Type II , Docetaxel , Neoadjuvant Therapy , Receptor, ErbB-2 , Trastuzumab , Humans , Female , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Middle Aged , Trastuzumab/administration & dosage , Trastuzumab/adverse effects , Trastuzumab/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/mortality , Breast Neoplasms/genetics , Receptor, ErbB-2/metabolism , Adult , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Docetaxel/administration & dosage , Docetaxel/adverse effects , Carboplatin/administration & dosage , Carboplatin/adverse effects , Aged , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/therapeutic use , Cyclophosphamide/administration & dosage , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Fluorouracil/therapeutic use , Poly-ADP-Ribose Binding Proteins/genetics , Anthracyclines/administration & dosage , Anthracyclines/therapeutic use , Epirubicin/administration & dosageABSTRACT
Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.
Subject(s)
Acrylamides , Aniline Compounds , Carcinoma, Non-Small-Cell Lung , DNA Topoisomerases, Type II , Drug Resistance, Neoplasm , ErbB Receptors , Lung Neoplasms , Topoisomerase II Inhibitors , Humans , Acrylamides/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/enzymology , Aniline Compounds/pharmacology , ErbB Receptors/genetics , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/enzymology , Lung Neoplasms/metabolism , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Cell Line, Tumor , Topoisomerase II Inhibitors/pharmacology , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Animals , Mice , Mutation , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Drug Synergism , DNA Damage , Piperazines/pharmacology , Etoposide/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. METHODS: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. RESULTS: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. CONCLUSION: Targeting the PRRX1-TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.