EMP3 negatively modulates breast cancer cell DNA replication, DNA damage repair, and stem-like properties.
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ABSTRACT:
SUBMITTER: Zhou K
PROVIDER: S-EPMC8435533 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The functional significance of the chemokine receptor CCR5 in human breast cancer epithelial cells is poorly understood. Here, we report that CCR5 expression in human breast cancer correlates with poor outcome. CCR5+ breast cancer epithelial cells formed mammospheres and initiated tumors with >60-fold greater efficiency in mice. Reintroduction of CCR5 expression into CCR5-negative breast cancer cells promoted tumor metastases and induced DNA repair gene expression and activity. CCR5 antagonists Maraviroc and Vicriviroc dramatically enhanced cell killing mediated by DNA-damaging chemotherapeutic agents. Single-cell analysis revealed CCR5 governs PI3K/Akt, ribosomal biogenesis, and cell survival signaling. As CCR5 augments DNA repair and is reexpressed selectively on cancerous, but not normal breast epithelial cells, CCR5 inhibitors may enhance the tumor-specific activities of DNA damage response-based treatments, allowing a dose reduction of standard chemotherapy and radiation.Significance: This study offers a preclinical rationale to reposition CCR5 inhibitors to improve the treatment of breast cancer, based on their ability to enhance the tumor-specific activities of DNA-damaging chemotherapies administered in that disease. Cancer Res; 78(7); 1657-71. ©2018 AACR.
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Project description:Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival.
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Project description:The activity of eukaryotic DNA polymerase delta (Pol ?) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol ? is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2.HEK293 cells were transfected with POLD1 expression plasmid or shRNA, cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells were analyzed.HEK293 cells were transfected with POLD1 expression plasmid or shRNA. POLD1 downregulation by shRNA suppressed cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells. However, POLD1 overexpression had no significant effects on these processes. Finally, comet assay showed that POLD1 downregulation led to increased DNA damage.Our results suggest that human POLD1 plays important role in the regulation of cell cycle progression and DNA damage repair.
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Project description:BackgroundCancer stem cells (CSCs) possess abilities of self-renewal and differentiation, have oncogenic potential and are regarded to be the source of cancer recurrence. However, the mechanism by which CSCs maintain their stemness remains largely unclear.MethodsIn this study, the cell line-derived ovarian CSCs (OCSCs), 3AO and Caov3, were enriched in serum-free medium (SFM). Differentially expressed proteins were compared between the OCSC subpopulation and parental cells using liquid chromatography (LC)-mass spectrometry (MS)/MS label-free quantitative proteomics. Sphere-forming ability assays, flow cytometry, quantitative real-time polymerase chain reaction (qPCR), western blotting, and in vivo xenograft experiments were performed to evaluate stemness. RNA-sequencing (RNA-seq) and pyrosequencing were used to reveal the mechanism by which STON2 negatively modulates the stem-like properties of ovarian cancer cells.ResultsAmong the 74 most differentially expressed proteins, stonin 2 (STON2) was confirmed to be down-regulated in the OCSC subpopulation. We show that STON2 negatively modulates the stem-like properties of ovarian cancer cells, which are characterized by sphere formation, a CD44+CD24- ratio, and by CSC- and epithelial mesenchymal transition (EMT)-related markers. STON2 knockdown also accelerated tumorigenesis in NOD/SCID mice. Further investigation revealed a downstream target, mucin 1 (MUC1), as up-regulated upon the down regulation of STON2. A decrease in both DNA methyltransferase 1 (DNMT1) expression and methylation in the promoter region of MUC1 was associated with subsequently elevated MUC1 expression, as detected in STON2 knockdown in 3AO and Caov3 cells. Direct DNMT1 knockdown simultaneously elevated MUC1 expression. The functional significance of this STON2-DNMT1/MUC1 pathway is supported by the observation that STON2 overexpression suppresses MUC1-induced sphere formation of OCSCs. The paired expression of STON2 and MUC1 in ovarian cancer specimens was also detected revealing the prognostic value of STON2 expression to be highly dependent on MUC1 expression.ConclusionsOur results imply that STON2 may negatively regulate stemness in ovarian cancer cells via DNMT1-MUC1 mediated epigenetic modification. STON2 is therefore involved in OCSC biology and may represent a therapeutic target for innovative treatments aimed at ovarian cancer eradication.
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Project description:The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity.
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Project description:Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.
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Project description:Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA-binding protein involved in chromatin remodeling, RNA processing and the DNA damage response. In addition, increased hnRNPK expression has been associated with tumor development and progression. A variety of post-translational modifications of hnRNPK have been identified and shown to regulate hnRNPK function, including phosphorylation, ubiquitination, sumoylation and methylation. However, the functional significance of hnRNPK arginine methylation remains unclear. In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKC?. Notably, the engineered U2OS cells carrying an Arg296/Arg299 methylation-defective hnRNPK mutant exhibited increased apoptosis upon DNA damage. While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part. Here, we provide the first evidence that the arginine methylation of hnRNPK negatively regulates cell apoptosis through PKC?-mediated signaling during DNA damage, which is essential for the anti-apoptotic role of hnRNPK in apoptosis and the evasion of apoptosis in cancer cells.
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Project description:Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after ?-irradiation (?-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and ?H2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-? and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.
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Project description:Replication origins are under tight regulation to ensure activation occurs only once per cell cycle [1, 2]. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint [2-7]. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions [5]. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks [3]. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control [8-10], to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs) [10-12]. Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation [13, 14]. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker ?H2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation.
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Project description:The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.
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