MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.

RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication.

Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

A Long Noncoding RNA Regulates Sister Chromatid Cohesion.

Long noncoding RNAs (lncRNAs) are involved in diverse cellular processes through multiple mechanisms. Here, we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), that is transcriptionally activated by MYC and is upregulated in multiple cancer types. The expression of CONCR is cell cycle regulated, and it is required for cell-cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication and sister chromatid cohesion. These findings unveil a direct role for an lncRNA in the establishment of sister chromatid cohesion by modulating DDX11 enzymatic activity.

WEE1 inhibitor and ataxia telangiectasia and RAD3-related inhibitor trigger stimulator of interferon gene-dependent immune response and enhance tumor treatment efficacy through programmed death-ligand 1 blockade.

WEE1 plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response (DDR). Inhibition of WEE1 can increase the instability of the genome and have anti-tumor effects in some solid tumors. However, it has certain limitations for multiple cancer cells from different lineages. Therefore, we consider the use of synthetic lethal interactions to enhance the therapeutic effect. Our experiments proved that WEE1 inhibitor (WEE1i) can activate the ataxia telangiectasia and RAD3-related (ATR) pathway and that blockage of ATR dramatically sensitized the WEE1i-induced cell death. The tumor-selective synthetic lethality between bioavailable WEE1 and ATR inhibitors led to tumor remission in vivo. Mechanistically, the combination promoted the accumulation of cytosolic double-strand DNA, which subsequently activated the stimulator of the interferon gene (STING) pathway and induced the production of type I interferon and CD8+ T cells, thereby inducing anti-tumor immunity. Furthermore, our study found that immune checkpoint programmed death-ligand 1 is upregulated by the combination therapy, and blocking PD-L1 further enhances the effect of the combination therapy. In summary, as an immunomodulator, the combination of WEE1i with ATR inhibitor (ATRi) and immune checkpoint blockers provides a potential new approach for cancer treatment.

UBE2C mRNA expression controlled by miR-300 and HuR determines its oncogenic role in gastric cancer.

Ubiquitin Conjugating Enzyme E2 C (UBE2C) has a key oncogenic role in many human malignancies, including gastric cancer. However, it remains largely unknow at which level UBE2C expression is altered, as well as what are the downstream targets of UBE2C. In this study, we show that UBE2C is frequently overexpressed in gastric cancer patients. Interestingly, high expression of UBE2C mRNA instead of genome amplification is the predominant alterations observed in both stomach adenocarcinoma. We then confirmed that silencing UBE2C not only suppresses gastric cancer colony formation, but also inhibits DNA biosynthesis. Furthermore, we discovered that microRNA-300 is able to suppress gastric cancer progression through reducing UBE2C mRNA abundance, which is protected by an RNA binding protein HuR. Lastly, through an analysis of genes whose expressions correlate with that of UBE2C from gastric cancer cell lines, we have proposed several key genes that can be regulated by UBE2C, contributing to its oncogenic activity.

EGF Conjugation Improves Safety and Uptake Efficacy of Titanium Dioxide Nanoparticles.

Titanium dioxide nanoparticles (TiO2 NPs) have a strong potential for cancer therapeutic and bioimaging applications such as photodynamic therapy (PDT) and photodynamic diagnosis (PDD). Our previous results have shown that TiO2 NPs have a low cellular uptake and can induce cell proliferation. This suggests that TiO2 NPs could increase the risk of tumor overgrowth while being used for PDD and PDT. To solve this problem, we constructed epidermal growth factor-ligated polyethylene glycol-coated TiO2 NPs (EGF-TiO2 PEG NPs). In this work, we studied the effect of EGF conjugation on the cellular uptake of TiO2 PEG NPs. Then, we investigated the effect of both non-conjugated and EGF-TiO2 PEG NPs on the A431 epidermal cancer cell line, proliferation and growth via the investigation of EGFR localization and expression. Our results indicated that TiO2 PEG NPs induced EGFRs aggregation on the A431 cells surface and induced cell proliferation. In addition, EGF-TiO2 PEG NPs induced the internalization of EGFRs inside of cells with increased cellular NPs uptake and decreased cellular proliferation compared to TiO2 PEG NPs-treated cells. These findings suggest that EGF conjugation can increase the efficacy of TiO2 PEG NPs for biomedical applications such as PDD and PDT with decreased risk of tumor overgrowth.

Pharmacological targeting of RAD6 enzyme-mediated translesion synthesis overcomes resistance to platinum-based drugs.

Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.

Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

Lipoxygenase Inhibitors Nordihydroguaiaretic Acid and Fungus Lecanicillum lecanii Extract Induce Death of Lymphoid Leukemia Cells.

We studied the effect of lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and fungus Lecanicillum lecanii extract on lymphatic leukemia P388 cells. The cells grown in the abdominal cavity of DBA2 mice for 7 days were transferred into a nutrient medium. The effect of lipoxygenase inhibitors was evaluated by changes in cell number, trypan blue staining, nucleus damage, and changes in cell distribution by DNA content after 22-h incubation. NDGA and fungus extract induced apoptotic death of lymphatic leukemia cells, which was seen from nucleus damage and reduced DNA content in cells. IC50 for NDGA and fungus extract was 0.66 and 5.5 µg/ml, respectively.

Effects of silibinin on growth and invasive properties of human ovarian carcinoma cells through suppression of heregulin/HER3 pathway.

Epithelial ovarian cancer (EOC) is the most fatal gynecological malignancy due to its high proliferative and invasive capacities. A heregulin (HRG)/HER3 autocrine loop increases proliferative and metastatic properties of EOC cells, suggesting that modulators of this signaling pathway may prove effective to trammel growth and motility of these cells. This study aimed to evaluate the effects of multi-tyrosine kinase inhibitor silibinin on proliferative and invasive characteristics of EOC cell lines OVCAR8 and SKOV3 through suppression of the HRG/HER3 pathway. To achieve this, the effects of silibinin on proliferation, DNA synthesis, clonogenicity, cell cycle progression, cathepsin B enzymatic activity, and migration and invasion were explored in vitro. Silibinin suppressed proliferation, DNA synthesis, and clonogenic abilities of OVCAR8 and SKOV3 cells through inhibition of the autocrine HRG/HER3 circuit. Silibinin-mediated attenuation of the HER3 signaling disabled the HER3/AKT/survivin axis and thereby, induced G1/S cell cycle arrest. Furthermore, silibinin reduced invasive potentials of the EOC cells through quelling the HRG/HER3 pathway and suppression of cathepsin B activity. Altogether, these results suggest that silibinin is a potential anti-cancer drug to inhibit proliferative and invasive characteristics of the EOC cells that exhibit an autocrine HRG/HER3 pathway.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses.

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

PI3K is required for both basal and LPA-induced DNA synthesis in oral carcinoma cells.

BACKGROUND: The glycerophospholipid lysophosphatidic acid (LPA), which is present in most tissues and in high concentrations in saliva, may exert profound effects on oral cancer cells. We have investigated mitogenic signalling induced by LPA in the two oral carcinoma cell lines, D2 and E10, focusing on the role of EGFR transactivation and downstream pathways. METHODS: Two oral squamous carcinoma cell lines, D2 and E10, were analysed for effects of LPA on signalling pathways and induction of DNA synthesis. Pathway activation was investigated by examining phosphorylation of signalling proteins and by the use of specific pathway inhibitors. RESULTS: The D2 cells had higher levels of activated signalling proteins and higher DNA synthesis activity in the basal condition than E10 cells. EGF did not induce proliferation in D2 cells, whereas LPA induced proliferation in both cell lines, by mechanisms depending on EGFR transactivation. Release of EGFR ligands was involved in basal and LPA-induced proliferation in both D2 and E10 cells. The proliferation in D2 cells was dependent on the PI3K/Akt pathway, but not the MEK/ERK pathway. In E10 cells, the PI3K/Akt, MEK/ERK and p38 pathways were all involved in the proliferation. CONCLUSION: Transactivation of EGFR is required for LPA-induced DNA synthesis in D2 and E10 cells. Our results also show that although proliferation of oral carcinoma cells is regulated by several pathways, and differentially in E10 and D2 cells, the PI3K pathway has a crucial role in both cell lines.

MicroRNAs and cell cycle of malignant glioma.

The control of malignant glioma cell cycle by microRNAs (miRNAs) is well established. The deregulation of miRNAs in glioma may contribute to tumor proliferation by directly targeting the critical cell-cycle regulators. Tumor suppressive miRNAs inhibit cell cycle through repressing the expression of positive cell-cycle regulators. However, oncogenic miRNAs promote the cell-cycle progression by targeting cell-cycle negative regulators. Recent studies have identified that transcription factors had involved in the expression of miRNAs. Transcription factors and miRNAs are implicated in regulatory network of glioma cell cycle, the deregulation of these transcription factors might be a cause of the deregulation of miRNAs. Abnormal versions of miRNAs have been implicated in the cell cycle of glioma. Based on those, miRNAs are excellent biomarker candidates and potential targets for therapeutic intervention in glioma.